Altered Dark- and Photoconversion of Phytochrome B Mediate Extreme Light Sensitivity and Loss of Photoreversibility of the phyB-401 Mutant E´ va A´da´ m1., Andrea Hussong2., Ja´ nos Bindics2, Florian Wu¨ st2, Andra´ s Viczia´ n1, Marcus Essing 2 , Ma´ tya´ s Medzihradszky2, Stefan Kircher2, Eberhard Scha¨ fer2,3, Ferenc Nagy1,4* 1 Institute of Plant Biology, Biological Research Centre, Szeged, Hungary, 2 Institut f u¨r Biologie II/Botanik, Universita¨t Freiburg, Freiburg, Germany, 3 Centre of Biological Signalling (BIOSS), Universita¨t Freiburg, Freiburg, Germany, 4 School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom Abstract The phyB-401 mutant is 103 fold more sensitive to red light than its wild-type analogue and shows loss of photoreversibility of hypocotyl growth inhibition. The phyB-401 photoreceptor displays normal spectral properties and shows almost no dark reversion when expressed in yeast cells. To gain insight into the molecular mechanism underlying this complex phenotype, we generated transgenic lines expressing the mutant and wild-type phyB in phyB-9 background. Analysis of these transgenic lines demonstrated that the mutant photoreceptor displays a reduced rate of dark-reversion but normal Pfr to Pr photoconversion in vivo and shows an altered pattern of association/dissociation with nuclear bodies compared to wild- type phyB. In addition we show (i) an enhanced responsiveness to far-red light for hypocotyl growth inhibition and CAB2 expression and (ii) that far-red light mediated photoreversibility of red light induced responses, including inhibition of hypocotyl growth, formation of nuclear bodies and induction of CAB2 expression is reduced in these transgenic lines. We hypothesize that the incomplete photoreversibility of signalling is due to the fact that far-red light induced photoconversion of the chromophore is at least partially uncoupled from the Pfr to Pr conformation change of the protein. It follows that the phyB-401 photoreceptor retains a Pfr-like structure (Pr*) for a few hours after the far-red light treatment. The greatly reduced rate of dark reversion and the formation of a biologically active Pr* conformer satisfactorily explain the complex phenotype of the phyB-401 mutant and suggest that amino acid residues surrounding the position 564 G play an important role in fine-tuning phyB signalling. Citation: A´da´m E´, Hussong A, Bindics J, W u¨st F, Viczia´n A, et al. (2011) Altered Dark- and Photoconversion of Phytochrome B Mediate Extreme Light Sensitivity and Loss of Photoreversibility of the phyB-401 Mutant. PLoS ONE 6(11): e27250. doi:10.1371/journal.pone.0027250 Editor: Frank G. Harmon, USDA-ARS, United States of America Received October 7, 2010; Accepted October 12, 2011; Published November 3, 2011 Copyright: 2011 A´ da´m et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Work in Freiburg was supported by a grant of the DFG (SFB592) to ES, FN, and SK; and a grant from FRSYS and BIOSS to ES. Work in Szeged was supported by a grant from OTKA (81399) to FN, and work in Edinburgh was supported by a SULSA professorial grant to FN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nagyf@brc.hu . These authors contributed equally to this work. Introduction Plants are sessile organisms, which have established a consid- erable plasticity of development to respond to changes in the natural environment. Light, a highly variable environmental factor is used not only as the main energy source, but also as an environmental cue to regulate plant growth and development. To monitor these changes, plants have evolved several classes of photoreceptors: the UV-B photoreceptors [1,2], the blue/UV-A sensing cryptochromes controlling plant development [3], the phototropins controlling directional growth, chloroplast re-orien- tation and stomata opening [4,5] and the red/far-red absorbing phytochromes controlling plant development [6,7,8]. In Arabidopsis thaliana phytochromes are encoded by a small gene family of five members, PHYA to PHYE [9]. The phytochromes in vivo exist as homo and/or heterodimers of about 125 kDa subunits [10] and each subunit contains a covalently linked, linear tetrapyrrole chromophore. The phytochromes are synthesized in their R light absorbing, biologically inactive Pr form in darkness. Absorption of R light converts them into their FR light absorbing biologically active Pfr form that again can be converted back into Pr form upon absorption of FR light [7]. In addition to photoconversion, the Pfr form can also fall back into the Pr form spontaneously, since the Pfr form has lower thermostability. This light-independent conformation change of the photoreceptor, also called dark reversion, was first suggested to function as an additional inactivation process by Eichenberg et al. [11]. It has been demonstrated by these authors that dark reversion beside being an intrinsic property of the molecule is also affected by cellular components and shows strong temperature dependence [12]. Whether the molecular dynamics of dark transition from Pfr to Pr are identical to and mediated by the same intermediate states as the light induced Pfr to Pr transition is not yet known. Based on their mode of action and the light stability of the proteins, phytochromes can be divided into two classes. Type I phytochromes controlling the Very Low Fluence Responses (VLFR) and the far-red High Irradiance Responses (HIR) show rapid degradation of the Pfr form. Type II phytochromes are light PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27250