Final Report Summary - PHYB (Identification and functional analysis of the sub-nuclear protein complex of phytochrome B photoreceptor in Arabidopsis thaliana)
Light is one of the most important environmental signals regulating a wide range of physiological and developmental processes in plants, from seed germination to flowering. To perceive changes in the light, plants evolved photoreceptors specialised in absorbing light of different wavelengths. Receptors for red and far-red light (620 - 750 nm) are called phytochromes. In Arabidopsis thaliana five phytochromes, PhyA-PhyE have been identified. PhyA and PhyB are the most prominent phytochromes to mediate photomorphogenesis under normal conditions. PhyA controls seed germination and seedling development while phyB has major role to control light responses in mature, de-etiolated green plants. Although the phytochromes were discovered many decades ago, our present knowledge about the exact number of components and molecular events required for phyB-mediated signalling cascade(s) is fairly limited. Hence, our research aimed for the better understanding of light sensing and PhyB signal transduction in plants. Recent studies showed that red and far-red light induced signal transduction is to a great extend mediated by nucleo-cytoplasmic partition of phytochromes. After light induction, all phytochromes translocate from the cytoplasm into the nucleus and form nuclear complexes called nuclear bodies (NBs) or photobodies. However, the proper function and the molecular composition of these nuclear photobodies is not known. The project aimed to purify the phyB containing NBs and to identify new factors involving NB formation and phyB mediated red-light signalling pathway.
To accomplish this goal we used a protein-based approach instead of time-consuming screening-based genetic approaches. To investigate the molecular composition of NBs we immunoprecipitated the phyB-green fluorescent protein (GFP) from transgenic Arabidopsis thaliana that expressed phyB:GFP driven by the 35S promoter. After immunopurification, samples were analysed by mass spectrometry. As result, 64 putative interacting proteins were identified. Based upon the putative function and the localisation we selected one protein for our further analysis. For our investigations, we generated transgenic Arabidopsis plants expressing the fluorescent tagged version of this protein either together with phyB-YFP or alone. Most importantly, we successfully showed that our candidate protein was in fact a a new factor in phyB-containing nuclear photobodies. First, the localisation of the candidate protein is strongly light dependent; it forms NBs in under red light but not in far-red or blue light, or in darkness. Second, these NBs require endogenous phyB, as it failed to form NBs in phyB null mutant plants. Third, these NBs perfectly overlap in localisation experiments with phyB photobodies indicating that the candidate protein forms photobodies together with phyB. According to our current knowledge, this is the first protein that has been shown in vivo as a photobody component.
In the second year of the project we aimed to determine the role of our candidate protein in phyB signalling at the physiological level. We generated ribonucleic acid interference (RNAi) lines to reduce the expression level of the candidate gene and overexpressing lines expressing the candidate gene in different genetic background, including wild-type, phyB null mutant, phyA null mutant and phyB overexpressing lines. The effect of the candidate protein has been examined by measuring the hypocotyl elongation of seedlings under red, far-red and blue light compared to the hypocotyl length of dark-grown etiolated seedlings. Interestingly, the overexpression of the candidate gene resulted in a mild but significant elongation of hypocotil in any light condition. Reciprocally, the reduced level of the candidate protein in the RNAi lines caused a hypersensitivity indicating the negative role of the protein in all light signalling pathway. However, the overexpression of phyB compensated the negative effect of the candidate protein under red light but not in blue light. Based on the observation that the candidate protein (i) is a component of nuclear photobodies in red light but not in blue or far-red light, (ii) co-localises and interacts with phyB under red light and (iii) acts as a negative factor of light signalling that can be compensated by phyB in red light but not in blue, we suggest that one function of the photobodies might be the sequestering of negative factors involved in light signalling.
During the project the results followed very well the objectives and allowed us to reveal a new component of photobodies. The examinations of the physiological role of this protein lead us to build a working hypothesis about the function of photobodies. The respective publication is in preparation. The researcher was also involved to work in collaboration with two Hungarian laboratory resulting one first author and two co-author publication and two more manuscripts are under preparation.
To accomplish this goal we used a protein-based approach instead of time-consuming screening-based genetic approaches. To investigate the molecular composition of NBs we immunoprecipitated the phyB-green fluorescent protein (GFP) from transgenic Arabidopsis thaliana that expressed phyB:GFP driven by the 35S promoter. After immunopurification, samples were analysed by mass spectrometry. As result, 64 putative interacting proteins were identified. Based upon the putative function and the localisation we selected one protein for our further analysis. For our investigations, we generated transgenic Arabidopsis plants expressing the fluorescent tagged version of this protein either together with phyB-YFP or alone. Most importantly, we successfully showed that our candidate protein was in fact a a new factor in phyB-containing nuclear photobodies. First, the localisation of the candidate protein is strongly light dependent; it forms NBs in under red light but not in far-red or blue light, or in darkness. Second, these NBs require endogenous phyB, as it failed to form NBs in phyB null mutant plants. Third, these NBs perfectly overlap in localisation experiments with phyB photobodies indicating that the candidate protein forms photobodies together with phyB. According to our current knowledge, this is the first protein that has been shown in vivo as a photobody component.
In the second year of the project we aimed to determine the role of our candidate protein in phyB signalling at the physiological level. We generated ribonucleic acid interference (RNAi) lines to reduce the expression level of the candidate gene and overexpressing lines expressing the candidate gene in different genetic background, including wild-type, phyB null mutant, phyA null mutant and phyB overexpressing lines. The effect of the candidate protein has been examined by measuring the hypocotyl elongation of seedlings under red, far-red and blue light compared to the hypocotyl length of dark-grown etiolated seedlings. Interestingly, the overexpression of the candidate gene resulted in a mild but significant elongation of hypocotil in any light condition. Reciprocally, the reduced level of the candidate protein in the RNAi lines caused a hypersensitivity indicating the negative role of the protein in all light signalling pathway. However, the overexpression of phyB compensated the negative effect of the candidate protein under red light but not in blue light. Based on the observation that the candidate protein (i) is a component of nuclear photobodies in red light but not in blue or far-red light, (ii) co-localises and interacts with phyB under red light and (iii) acts as a negative factor of light signalling that can be compensated by phyB in red light but not in blue, we suggest that one function of the photobodies might be the sequestering of negative factors involved in light signalling.
During the project the results followed very well the objectives and allowed us to reveal a new component of photobodies. The examinations of the physiological role of this protein lead us to build a working hypothesis about the function of photobodies. The respective publication is in preparation. The researcher was also involved to work in collaboration with two Hungarian laboratory resulting one first author and two co-author publication and two more manuscripts are under preparation.