Using natural variation for the investigation of the phytochrome A sequence/function relationship
Final Report Abstract
Phytochrome A (phyA) is an important photoreceptor in plants that plays an essential role for seedling establishment in deep canopy shade where the available light spectrum is dominated by far-red light. PhyA also enables plants to sense and respond to very weak light or short light stimuli such as during soil cultivation. In the project, we screened the 1001 Genomes collection for accessions that are less sensitive to far-red light, suggesting that either phyA is less active in these accessions or phyA downstream signalling is less efficient compared to the reference accession Col-0. Most accessions that are less sensitive to far-red light than Col-0 do not contain polymorphisms in the PHYA gene that lead to amino acid changes in the phyA protein, indicating that possibly phyA downstream signalling is affected in these accessions. For accessions in which the hyposensitivity to far-red light segregates as monogenic trait when crossed into Col-0 background, we plan to identify the polymorphisms responsible for the phenotype either by an educated guess approach or by mapping. We also identified a total of 21 accessions that contain amino acid substitutions in the phyA protein and are much less sensitive to far-red light than the Col-0 reference accession. In future work, we want to investigate if and how these amino acid substitutions affect the spectral and molecular properties of phyA. According to the schedule, these analyses should have been carried out as part of the project. However, due to limited access to the lab and the delayed delivery of essential chemicals and kits during the Corona pandemic, this part of the project was delayed. In the second part of the project, we optimised a protocol for the expression and purification of tag-free photoactive phyA using an E. coli strain engineered to synthesise the natural chromophore of phyA. This part was successful and we established a protocol that allowed us to obtain enough phyA of sufficiently high purity to start crystallisation trials. We identified conditions under which we obtain diffracting phyA protein crystals. The longterm goal is to build on this protocol and further improve the procedure to obtain crystal structures of different phyA variants that could provide insight into how amino acid substitutions in phyA affect its function. Finally, the protocol established for the expression of photoactive phyA in E. coli and its subsequent purification also lead to great progress in other projects and contributed to a publication showing that phyA regulates the association of specific transcription factors with target promoters to regulate seed germination.
Publications
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The phytochrome interacting proteins ERF55 and ERF58 repress light-induced seed germination in Arabidopsis thaliana. Nature Communications, 13(1).
Li, Zenglin; Sheerin, David J.; von Roepenack-Lahaye, Edda; Stahl, Mark & Hiltbrunner, Andreas
