Significance and timeliness: Photosynthetic organisms face rapid fluctuations in light intensity. High light is harmful, resulting in reduction in plant productivity . The most light-sensitive component is the Photosystem II (PSII) reaction center (RCII). However, a mechanism exists that protects RCII against excess light via a dissipation of absorbed energy into heat (NPQ) . Genetic manipulation of plants with optimized NPQ is of paramount importance for crop improvement and mitigation of the threat of global warming . The key protein involved in NPQ is called PsbS . By sensing the proton gradient it binds to the PSII antenna causing its aggregation and switching into the photoprotective state . A similar process exists in diatom algae and is potentiated by the LHCX1 protein that causes extremely large NPQ (super-NPQ) . The aim of this project is to express LHCX1 protein in Arabidopsis in order to test the hypothesis of convergent evolution of protective proteins in plants and diatoms as well as to attain superprotective plants, capable of tolerating extremes of light.
Project core: The Ruban laboratory is the world leading group that works on protection of the photosynthetic apparatus against abiotic stresses. Now, it is essential to involve genetic manipulation in order to improve this stress tolerance in plants. The Hanke group have extensive expertise in transgenic manipulation of Arabidopsis plants to introduce heterologous proteins, and target them to the chloroplast [6,7]. Arabidopsis mutants of PsbS will be transformed with the following constructs, to generate the genetic resources necessary to answer these research questions:
1st Construct, the gene for the diatom LHCX1 protein. This will test whether LHCX1 can act as a functional homologue of PsbS, and establish whether LHCX1 can act as the basis for “superquenching”.
2nd Constructs, the gene for LHCX1, mutated at specific residues thought to be important for the functional mechanism. This will test hypotheses about LHCX1 function.
3rd Constructs, sequences for the native Arabidopsis PsbS protein, including mutations to introduce LHCX1 like features. This will test the structural basis of the “superquenching” mechanism.
Genes will be introduced on a disabled transposable element vector via Agrobacterial floral dip of the Arabidopsis PsbS mutants. Expression of constructs will be driven by the native PsbS promoter, and selection performed via glyphosate resistance. The 1st construct is already prepared and need only be sub-cloned into the plant transformation vector prior to transformation.
Electron microscopy technique, freeze-fracture, will be employed to visualize complexes of PSII in the created plants in order relate likely structural alterations caused by the introduction of LHCX1 to alterations in NPQ parameters – amplitude and kinetics of high light light-induced formation and relaxation in the dark. Biochemical techniques such as isoelectric focusing, gel filtration and ultracentrifugation will be employed in order to isolate the complete spectrum of light harvesting antenna complexes and detect possible compensatory changes in their composition in the constructed plants and the localisation of the LHCX1 protein.
The studentship will cover tuition fees and provide an annual tax-free maintenance allowance for 3 years at the Research Council rate (£16,777 in 2018/19).
Applicants must meet the entry requirements for our PhD programmes. A masters degree in plant molecular biology and biochemistry is desirable.
Informal enquiries about the project can be sent to Professor Alexander Ruban (firstname.lastname@example.org). Formal applications should be submitted online by the stated deadline.