Temperature Sensitive Photosynthesis: Point Mutated CEF-G, PRK, or PsbO Act as Temperature-Controlled Switches for Essential Photosynthetic Processes

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.

Structure of Photosystem I Supercomplex Isolated from a Chlamydomonas reinhardtii Cytochrome b6f Temperature-Sensitive Mutant

“…In this study, we used a selection system designed to identify mutations in the photosystems (see Section 2 ), as described elsewhere [ 43 ]. When exposed to light, the WT strain of C. reinhardtii produces biomass and chemical energy.…”

“…A pool of randomly mutated strains of the model unicellular photosynthetic organism C. reinhardtii was produced using ultraviolet (UV) irradiation (Baker company, Sanford, ME, USA), as described previously [ 43 ]. UV irradiation of C. reinhardtii caused random mutagenesis with a 90% mortality rate ( Figure S1 and detailed analysis in the Supplementary Materials ).…”

“…To eliminate mutations in their genome that are unrelated to temperature sensitivity, we backcrossed the newly selected mutant strains with the WT, as described elsewhere [ 43 , 45 ]. We screened the progeny again as before, and two daughter cells showed the phenotype out of each tetrad obtained.…”

“…A TSP mutant allows the induction and maintenance of anaerobic conditions in light simply by controlling the culture temperature. A screening method has been developed, and thorough phenotyping of C. reinhardtii TSP mutants has enabled the selection of TSP1, TSP2, TSP3, and TSP4, according to a variety of criteria [ 43 ]. We applied these phenotyping methods in this study and used TSP4 as an additional control.…”

Dimeric and high-resolution structures of Chlamydomonas Photosystem I from a temperature-sensitive Photosystem II mutant

“…TSP4 has been demonstrated to completely degrade PSII upon a high-temperature treatment. The identified mutation on the PsbO gene caused a single amino acid change Pro101His in PsbO, which resulted in a temperature-sensitive PSII 12 . The elevated temperature resulted in the release of the mutated PsbO from the PSII complex and caused the entire complex to destabilise and degrade.…”