Green light control of plant form and function

Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S225–S233

P2.7 Structure-function analysis of phototropin blue-light receptors J. Christie, M. Jones, S. Sullivan, C. Thomson, (University of Glasgow, United Kingdom) Phototropins (phot1 and phot2) are blue light-activated serine/ threonine protein kinases that elicit a variety of photoresponses in plants. Light sensing by the phototropins is mediated by two flavin mononucleotide (FMN)-binding domains designated LOV1 and LOV2, located in the N-terminal region of the protein. Exposure to light results in the formation of a covalent adduct between the FMN chromophore and a conserved cysteine residue within the LOV domain. LOV2 photoexcitation is essential for phot1 function in Arabidopsis, and is necessary to activate phot1 kinase activity through lightinduced structural changes within a conserved Éø-helix situated C-terminal to LOV2. We have used site-directed mutagenesis to identify further amino acid residues that are important for phot1 activation by light. Mutagenesis of bacterially expressed LOV2 and full-length phot1 expressed in insect cells indicates that perturbation of the conserved salt bridge on the surface of LOV2 does not play a role in receptor activation. However, mutation of a conserved glutamine residue (Gln575) within LOV2, reported previously to be required to propagate structural changes at the LOV2 surface, attenuates light-induced autophosphorylation of phot1 expressed in insect cells without compromising FMN-binding. These findings, in combination with double mutant analyses, indicate that Gln575 plays an important role in coupling light-driven cysteinyl adduct formation from within LOV2 to structural changes at the LOV2 surface that lead to activation of the C-terminal kinase domain. Additional structure-function analysis of Arabidopsis phot1 will also be presented. doi:10.1016/j.cbpa.2007.01.499

P2.8 A base-catalyzed mechanism for dark state recovery in the Avena sativa phototropin-1 LOV2 domain J. Kennis, M. Alexandre, J. Arents, R. van Grondelle, (Vrije Universiteit (Vrije University), The Netherlands); K. Hellingwerf, (University of Amsterdam, The Netherlands) Phototropins are autophosphorylating serine/threonine kinases responsible for blue-light perception in plants; their action gives rise to phototropism, chloroplast relocation, and opening of stomatal guard cells. The kinase domain constitutes the Cterminal part of Avena sativa phototropin 1. The N-terminal part contains two Light, Oxygen or Voltage (LOV) sensing domains, LOV1 and LOV2, each bind a flavin mononucleotide (FMN) chromophore (?max = 447 nm, referred as D447) and form the light-sensitive domains, of which LOV2 is principal. Blue-light absorption produces a covalent adduct between a very

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conserved nearby cysteine residue and the C(4a) atom of the FMN moiety via the triplet state of the flavin. The covalent adduct thermally decays to regenerate the D447 dark state, with a rate that may vary several orders of magnitude between different species. We report that the imidazole base can act as a very efficient enhancer of the dark recovery of A. sativa phot1 LOV2 (AsLOV2) and some other well characterised LOV domains. Imidazole accelerates the thermal decay of AsLOV2 by three orders of magnitude in the sub-molar concentration range, via a base-catalysed mechanism involving base abstraction of the FMN N(5)-H adduct state and subsequent reprotonation of the reactive cystein. The LOV2 crystal structure suggests that the imidazole molecules may act from a cavity located in the vicinity of the FMN, explaining its high efficiency, populated through a channel connecting the cavity to the protein surface. Use of pH titration and chemical inactivation by diethylpyrocarbonate (DEPC) suggests that histidines located at the surface of the LOV domain act as base catalysts via an as yet unidentified H-bond network, operating at a rate of (55 s)−1 at pH 8. In addition, molecular processes other than histidine-mediated base catalysis contribute significantly to the total thermal decay rate of the adduct and operate at a rate constant of (65 s)−1, leading to a net adduct decay time constant of 30 s at pH 8. doi:10.1016/j.cbpa.2007.01.500

P2.9 Green light control of plant form and function K. Folta, S. Maruhnich, A. Dhingra, D. Kumar, (University of Florida, United States) Plants rely on signals from the environment to shape their growth and development. Of the suite of signals sensed, light influences both acute and long-term biological processes. Various aspects of the light signal, such as quantity (fluence and/or fluence rate), quality (wavelength) and duration (photoperiod) are integrated to generate appropriate biological response to best match ambient conditions. Light signals are sensed via a series of photosensors and integrated by their attendant signaling pathways. Of these, the phytochromes, cryptochromes, phototropins and other LOV-domain proteins have been well described, and are activated to wavebands similar to those that optimally stimulate photosynthesis. However, other wavelengths (such as far-red) certainly contain information that dramatically remodels gene expression and plant stature. Similarly, recent efforts have expanded on reports from the classical literature and examined the role of green light (500– 550 nm). Through the use of homemade narrow-bandwidth LED light arrays we have shown that green light induces a series of responses that are contrary to those typically associated with normal photomorphogenic development. Whereas red, blue and far-red light cause a decrease in stem elongation rate, green light induces an increase in stem elongation. Green light irradiation causes an acute down-regulation of specific chloroplast

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Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S225–S233

transcripts in the etiolated seedling, most of which are typically up-regulated by light. Green effects are apparent in a background of constant blue and red, although they are most salient at low fluence rates. In addition to their antagonistic course, the greenlight-specific phenomena do not occur in response to in blue, red or far-red light and persist in all photomorphogenic mutants tested, suggesting that they are mediated by a novel receptor. Taken together, the data indicate that green wavebands provide important information that tailors plant form and gene expression to best exploit a given light environment. doi:10.1016/j.cbpa.2007.01.501

P2.10 Mechanisms of response to ultraviolet-b radiation — A whole plant perspective J. Wargent, N. Paul, (Lancaster University, United Kingdom) Plant responses to ultraviolet (UV) radiation are numerous, resulting in rapid and permanent alteration to numerous aspects of plant form, physiology and biochemistry. Such responses to UV and UV-B (290–320 nm) radiation in particular have been previously studied largely due to concerns over ozone depletion, but a recent refocus towards the effects of environmentally relevant UV-B doses has provided an opportunity to investigate the mechanistic basis for several well defined plant responses. We have characterised some of these underlying mechanisms using a combination of crop species and Arabidopsis mutants, in an effort to build up a model of whole-plant response. (1) The role of UV in regulating leaf growth has been investigated using Lactuca sativa as a model species in a top-down approach, whereby leaf expansion rate, leaf biophysical properties, cell growth and cell wall peroxidase content have been subject to UV-B mediated change. (2) Upregulation of secondary metabolite biosynthesis has also been characterised in L. sativa, with UV absorbing compounds increased according to UV-B, plus leaf anthocyanin (pigment compound) concentration elevated by 60% in plants exposed to the highest UV dose compared to control. (3) The importance of various key pathways to the UV whole plant response are also currently under investigation with a selection of A. thaliana mutants. Such findings will not only add to the understanding of fundamental photobiological responses, but are also currently being successfully integrated into sustainable agronomic practice. doi:10.1016/j.cbpa.2007.01.502

P2.11 UVR8 defines a photomorphogenic UV-B-specific signalling pathway that regulates transcription G. Jenkins, B. Brown, C. Cloix, L. Headland, P. Herzyk, E. Kaiserli, (University of Glasgow, United Kingdom)

Plants are constantly exposed to ultraviolet-B (280–320 nm) radiation in sunlight. UV-B has the potential to cause damage to macromolecules and consequently plants need to protect against and repair UV damage to survive. High fluence rates of UV-B elicit stress responses via signalling pathways that overlap with defence and wound signalling pathways. In contrast, low fluence rates of UV-B stimulate the expression of genes involved in UV-protective responses. Hence the low fluence ‘photomorphogenic’ UV-B signalling pathway(s) promotes plant survival in UV-B. The aim of our research is to understand the processes of photomorphogenic UV-B perception and signal transduction that regulate transcription. We reported that Arabidopsis UV resistance locus 8 (UVR8) is a UV-B-specific signalling component that orchestrates expression of a range of genes with vital UV-protective functions (Brown et al., 2005, PNAS 102, 18225-30). We isolated several UVR8 mutants that are deficient specifically in the UV-B induction of gene expression and are highly susceptible to damage by UV-B. We found that UVR8 regulates expression of the transcription factor HY5 specifically in response to UV-B. HY5 is a key effector of the UVR8 pathway and is required for survival in UV-B. UVR8 is similar in sequence to the eukaryotic guanine nucleotide exchange factor RCC1, but differs in both activity and function. However, UVR8, like RCC1 is located principally in the nucleus and binds to chromatin via histones. Chromatin immunoprecipitation showed that UVR8 associates with chromatin in the HY5 promoter region, providing a mechanistic basis for its involvement in regulating transcription. Thus, UVR8 defines a new photomorphogenic signalling pathway in plants that plays a key role in regulating transcription specifically in response to UV-B. Recently we have extended our research to examine how UV-B regulates UVR8, in particular its subcellular localisation, and to define the role of the HYH transcription factor in UV-B responses. These findings will be presented. doi:10.1016/j.cbpa.2007.01.503

P2.12 SPA proteins are key repressors in light signal transduction and photoperiodic flowering U. Hoecker, (University of Cologne, Germany) The four-member SPA protein family of Arabidopsis functions in concert with the E3 ubiquitin ligase COP1 to suppress photomorphogenesis in dark-grown seedlings. Moreover, SPA proteins are essential for photoperiodic flowering. Mutations in SPA1 cause early flowering under short day but not long day conditions, and this phenotype is enhanced by additional loss of SPA3 and SPA4 function. These SPA1 SPA3 SPA4 triple mutants flower at the same time in long day and short day, indicating that the SPA gene family is essential for the inhibition of flowering under non-inductive short day. Early flowering of SPA1 mutants in short day is fully dependent on the floral activator