Abstract
Phototropins are plasma membrane-localized UVA/blue light photoreceptors which mediate phototropism, inhibition of primary hypocotyl elongation, leaf positioning, chloroplast movements, and stomatal opening. Blue light irradiation activates the C-terminal serine/threonine kinase domain of phototropin which autophosphorylates the receptor. Arabidopsis thaliana encodes two phototropins, phot1 and phot2. In response to blue light, phot1 moves from the plasma membrane into the cytosol and phot2 translocates to the Golgi complex. In this study the molecular mechanism and route of blue-light-induced phot2 trafficking are demonstrated. It is shown that Atphot2 behaves in a similar manner when expressed transiently under 35S or its native promoter. The phot2 kinase domain but not blue-light-mediated autophosphorylation is required for the receptor translocation. Using co-localization and western blotting, the receptor was shown to move from the cytoplasm to the Golgi complex, and then to the post-Golgi structures. The results were confirmed by brefeldin A (an inhibitor of the secretory pathway) which disrupted phot2 trafficking. An association was observed between phot2 and the light chain2 of clathrin via bimolecular fluorescence complementation. The fluorescence was observed at the plasma membrane. The results were confirmed using co-immunoprecipitation. However, tyrphostin23 (an inhibitor of clathrin-mediated endocytosis) and wortmannin (a suppressor of receptor endocytosis) were not able to block phot2 trafficking, indicating no involvement of receptor endocytosis in the formation of phot2 punctuate structures. Protein turnover studies indicated that the receptor was continuously degraded in both darkness and blue light. The degradation of phot2 proceeded via a transport route different from translocation to the Golgi complex.
Highlights
Many cellular processes in plants depend on sensing and responding to light direction, duration, quantity, and quality
The occurrence of cytosolic signal could be interpreted as soluble cytoplasmic phot2 or the receptor being present in intracellular compartment(s), for example the endoplasmic reticulum (ER)
The antibody used against phot2 was custom made by Agrisera and it is highly specific for Arabidopsis
Summary
Many cellular processes in plants depend on sensing and responding to light direction, duration, quantity, and quality. Different classes of photoreceptors have been identified that activate the light-dependent responses in an efficient and accurate manner. One of them is phototropin, a UVA/blue light receptor involved in regulating processes that are important for the promotion of plant growth and for the optimization of photosynthetic efficiency (Briggs, 2001). In the absence of blue light, LOV domains non-covalently bind flavin mononucleotide (FMN) as a chromophore. Blue light absorption results in the formation of a covalent adduct between the LOV domain and FMN, activating the C-terminal kinase domain which autophosphorylates phototropin (Pfeifer et al, 2010). The two phototropins in Arabidopsis, phot and phot, regulate various responses including phototropism (hypocotyl bending), stomatal movements, chloroplast redistribution, the inhibition of primary hypocotyl elongation, leaf positioning, and the stability of resistance protein HRT (Briggs and Christie, 2002; Folta et al, 2003; Inoue et al, 2008; Jeong et al, 2010). The signal transduction from phototropins involves secondary messengers, Ca2+, and phosphoinositides (Harada and Shimazaki, 2007; Chen et al, 2008; Aggarwal et al, 2013)
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