Erschienen in: The Plant Journal ; 77 (2014), 3. - S. 393-403 https://dx.doi.org/10.1111/tpj.12395 Reduced phototropism in pks mutants may be due to altered auxin-regulated gene expression or reduced lateral auxin transport Chitose Kami1,†, Laure Allenbach1, Melina Zourelidou2, Karin Ljung3,Fred eric Schutz€ 4, Erika Isono2, Masaaki K. Watahiki5, Kotaro T. Yamamoto5, Claus Schwechheimer2 and Christian Fankhauser1,* 1Center for Integrative Genomics, Faculty of Biology and Medicine, University of Lausanne, Genopode Building, 1015 Lausanne, Switzerland, 2Department of Plant Systems Biology, Technische Universitat Munchen, 85354 Freising-Weihenstephan, Germany, 3Umea˚ Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Umea˚ , Sweden, 4SIB, Swiss Institute of Bioinformatics, Genopode Building, 1015 Lausanne, Switzerland, and 5Division of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan *For correspondence (e mail [email protected]). †Present address: Botanical Gardens, Graduate School of Science, Osaka City University, 2000 Kisaichi, Katano shi, Osaka 576 0004, Japan. SUMMARY Phototropism allows plants to orient their photosynthetic organs towards the light. In Arabidopsis, pho- totropins 1 and 2 sense directional blue light such that phot1 triggers phototropism in response to low fluence rates, while both phot1 and phot2 mediate this response under higher light conditions. Phototro- pism results from asymmetric growth in the hypocotyl elongation zone that depends on an auxin gradi- ent across the embryonic stem. How phototropin activation leads to this growth response is still poorly understood. Members of the phytochrome kinase substrate (PKS) family may act early in this pathway, because PKS1, PKS2 and PKS4 are needed for a normal phototropic response and they associate with phot1 in vivo. Here we show that PKS proteins are needed both for phot1- and phot2-mediated phototro- pism. The phototropic response is conditioned by the developmental asymmetry of dicotyledonous seedlings, such that there is a faster growth reorientation when cotyledons face away from the light compared with seedlings whose cotyledons face the light. The molecular basis for this developmental effect on phototropism is unknown; here we show that PKS proteins play a role at the interface between development and phototropism. Moreover, we present evidence for a role of PKS genes in hypocotyl gravi-reorientation that is independent of photoreceptors. pks mutants have normal levels of auxin and normal polar auxin transport, however they show altered expression patterns of auxin marker genes. This situation suggests that PKS proteins are involved in auxin signaling and/or lateral auxin redistribution. Keywords: phototropism, phytochrome kinase substrate, phototropin 1, auxin, Arabidopsis thaliana. INTRODUCTION As sessile photoautotrophic organisms, plants need to 2010; Rizzini et al., 2011). These photoreceptors modulate adapt their growth constantly as physiology and develop- plant growth and development from seed germination until ment in an environment of fluctuating light conditions both senescence by control of the timing of key developmental have particularly strong influences on the plant’s entire life transitions and initiation of important adaptations (e.g. pho- cycle. Higher plants sense such changes with multiple totropism, shade avoidance; Christie, 2007; Franklin and photoreceptors that include a UV-B sensor, red and far-red Quail, 2010; Kami et al., 2010). Light sensing by the photot- photoreceptors of the phytochrome class and three distinct ropins (phot1 and phot2 in Arabidopsis thaliana) allows the families of specific blue-light receptors namely the crypto- optimization of photosynthetic activity by control of a range chromes, phototropins and ZTL/FKF1/LKP2 (Kami et al., of physiological responses that include phototropism, leaf 393 Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-398856 394 positioning, leaf flattening, chloroplast movements and and NPH3 (Lariguet et al., 2006; de Carbonnel et al., 2010; opening of stomata (Christie, 2007). Demarsy et al., 2012). PKS4 is phosphorylated by phot1 Phototropins are light-activated Ser/Thr protein kinases within seconds of blue-light perception (Demarsy et al., that are composed of an amino-terminal photosensory 2012). These findings, taken together with the strong domain and a carboxy-terminal protein kinase domain genetic interactions between nph3 and pks2 (de Carbonnel (Christie, 2007; Tokutomi et al., 2008). Two light oxygen et al., 2010), suggest that PKS proteins may also act early voltage (LOV) domains, LOV1 and LOV2, that each bind an in phototropin signaling, however the mechanism by FMN chromophore, compose the light-sensing portion of which they control phototropism remains unknown. An the photoreceptor with LOV2 playing a particularly additional complication comes from the role that PKS pro- important role (Christie et al., 2002; Cho et al., 2007). Upon teins also play in phytochrome signaling (Lariguet et al., light perception the protein kinase domain is liberated 2003; Schepens et al., 2008). Importantly, phytochromes, from the inhibitory activity of the photosensory domain and in particular phyA, enhance phototropism; this after a suite of light-induced conformational changes enhancement may depend, at least in part, on phyA-medi- (Harper et al., 2003; Matsuoka and Tokutomi, 2005; ated induction of PKS1 expression (Lariguet et al., 2006; Tokutomi et al., 2008). In Arabidopsis, several blue-light- Kami et al., 2012). induced phosphorylation sites have been identified in To obtain further insight into the role of PKS genes in phot1 and phot2 and it has been shown that phosphoryla- phototropism, we characterized the phototropic response tion in the activation loop of the protein kinase domain of of pks1, pks2 and pks4 single, double and triple mutants these proteins is essential for all tested physiological that had been grown under different blue-light intensities. responses (Inoue et al., 2008a, 2011; Sullivan et al., 2008). Using higher order mutants between pks and phot or phyA However, surprisingly, little information is known about we showed genetically that PKS proteins primarily act in the substrates of the phototropins (Christie et al., 2011; phot1 signaling. Tropic responses of the hypocotyl are Demarsy et al., 2012). developmentally modulated, as the orientation of the coty- Signal transduction events that occur upon phototropin ledons relative to the incoming light influences the activation depend, at least partially, upon the physiological response (Khurana et al., 1989). Our analysis shows that response because several phototropin signaling elements PKSs are important for this developmental regulation of are only required for a subset of phot-mediated responses the tropic response. Mechanistically, we show that PKS (Inada et al., 2004; Inoue et al., 2008b; de Carbonnel et al., proteins are dispensable for plasma membrane localization 2010). During phototropism, NPH3 has been shown to play of phot1. Auxin levels and polar auxin transport are normal a particularly important function, as a loss-of-function in pks1pks2pks4 seedlings, however auxin-regulated gene mutant was found to be aphototropic under all tested con- expression in the etiolated hypocotyl hook region is altered ditions (Motchoulski and Liscum, 1999). NPH3 and phot1 in pks1pks2pks4. We propose that PKS proteins act early in are plasma membrane-associated proteins that interact phot1 signaling by modulation of auxin signaling and/or with each other (Motchoulski and Liscum, 1999; Sakamoto lateral transport. and Briggs, 2002; Lariguet et al., 2006). In rice CPT1, the or- RESULTS tholog of NPH3, is essential for phototropism and acts upstream of lateral auxin redistribution that is needed for a In a previous study, we showed that pks1, pks2 and pks4 phototropic response (Haga et al., 2005; Esmon et al., are required for a normal phototropic response (Lariguet 2006). The interaction between phot1 and NPH3 and its et al., 2006). Our previous results suggested that PKS requirement upstream of auxin redistribution indicate that proteins acted in the phot1 rather than the phyA pathway NPH3 acts early during phototropism signaling. NPH3 is in the control of phototropism, but this action was not rapidly dephosphorylated in response to blue light in a demonstrated formally. In order to do so we first analyzed phot1-dependent manner (Pedmale and Liscum, 2007), phototropism in response to different fluence rates of moreover it is required for the down-regulation of phot1 unilateral blue light using long-term phototropic assays protein in daylight (Roberts et al., 2011). Both auxin trans- (Lariguet and Fankhauser, 2004). Consistent with the port and auxin signaling are required downstream of these results from previous experiments, phyA mutants showed early events (Tatematsu et al., 2004; Stone et al., 2008; a reduced phototropic response that was most apparent Christie et al., 2011; Ding et al., 2011; Willige et al., 2013). under low fluence rates (0.1 lmoles mÀ2 secÀ1 and below), Members of the phytochrome kinase substrate (PKS) while phot1 was defective at all fluence rates tested (Figure family (PKS1 PKS4 in Arabidopsis) play a role in a subset S1; Kami et al., 2012). In agreement with the proposal that of phototropin-mediated responses (Lariguet et al.,