Characterisation of mutants involved in Phytochrome A nuclear import and signal transduction

In the present work, an Ethyl Methane Sulphonate (EMS) mutagenesis screen was performed using a transgenic line expressing phyA: GFP fusion protein. Aim of the screen was to isolate and characterise Phytochrome A (phyA) nuclear transport mutants. The screening was performed in two steps. First, the mutagenised seedlings were selected on the basis of differences in hypocotyl length. In a second step, the selected putants were screened for altered localisation of phyA: GFP. The mutagenesis screen produced 47 mutants, of which 5 mutants were altered in the nuclear transport of phyA. These five mutants were further characterised into constitutive photomorphogenic phenotype (2 mutants), hypersensitive phenotype (1 mutant) and hyposensitive phenotype (2 mutants). Out of the two hyposensitive mutants, one was characterised in detail. It turned out to be a photoreceptor mutant. This mutant contained a single point mutation in the core region nucleotide at 2066 bp of the photoreceptor PHYA: GFP leading to an amino acid change from glutamate (E) to gycine (G). The mutation caused shift of the action spectrum to the red light ( 657 nm) in addition to the normal action peak in the far-red light (730 nm). The mutated photoreceptor was stable and was not degraded in red light and continuous white light. The mutated photoreceptor was constitutively localised in the nucleus even in darkness. The mutation also caused reduced abundance of the photoreceptor in darkness. Additionally it caused the presence of the mutated photoreceptor in different growth stages of Arabidopsis thaliana i.e. during embryogenesis, dry seeds and adult plants, which was in contrast to the wild type phyA. In the present work, mutant screen was performed under continuous RG9 light (756 nm) for the first time, with an intension to find mutants with shifted spectral sensitivity towards RG9 light. The screen also resulted in mutants that did not show altered localisation of phyA: GFP. In this category 3 hyposensitive and 7 hypersensitive mutants were characterised. The 3 hyposensitive mutants showed altered expression level and degradation of the endogenous phyA. Five of the 7 hypersensitive mutants showed hypersensitivity towards RG9 light. One of these mutants showed extreme hypersensitivity only in RG9 light as compared with far-red light. This mutant seems to be shifted in its sensitivity towards RG9 light. One of the 5 hypersensitive mutants isolated under RG9 screening was characterised in detail. Rough mapping analysis showed that the mutation locus was located to the upper arm of chromosome 4. The locus does not contain any known genes involved in light signalling. Fine mapping can identify a new gene involved in phyA signal transduction. In this study the primary nuclear transport mutant, which was characterised in detail, turned out to be a photoreceptor mutant. This suggests that the nuclear transport of the photoreceptor can be regulated by itself. But it is still possible that one of the four nuclear transport mutants which are yet to characterised in detail, contains mutation in signal transduction components involved in nuclear transport of phyA.

Author dc:creator

• Panigrahy, Madhusmita

Contributors dc:contributor

• Schäfer, Eberhard

• Kernimport
• Arabidopsis thaliana
• Licht
• nuclear import
• Phytochrome A
• signaltransduction
• mutant

Repository record source_url

https://freidok.uni-freiburg.de/data/1543

OAI identifier oai:identifier

oai:freidok.uni-freiburg.de:1543