| Abstract: | In Arabidopsis (Arabidopsis thaliana), the blue light photoreceptor phototropins (phot1 and phot2) fine-tune the photosynthetic status of the plant by controlling several important adaptive processes in response to environmental light variations. These processes include stem and petiole phototropism (leaf positioning), leaf flattening, stomatal opening, and chloroplast movements. The PHYTOCHROME KINASE SUBSTRATE (PKS) protein family comprises four members in Arabidopsis (PKS1–PKS4). PKS1 is a novel phot1 signaling element during phototropism, as it interacts with phot1 and the important signaling element NONPHOTOTROPIC HYPOCOTYL3 (NPH3) and is required for normal phot1-mediated phototropism. In this study, we have analyzed more globally the role of three PKS members (PKS1, PKS2, and PKS4). Systematic analysis of mutants reveals that PKS2 (and to a lesser extent PKS1) act in the same subset of phototropin-controlled responses as NPH3, namely leaf flattening and positioning. PKS1, PKS2, and NPH3 coimmunoprecipitate with both phot1-green fluorescent protein and phot2-green fluorescent protein in leaf extracts. Genetic experiments position PKS2 within phot1 and phot2 pathways controlling leaf positioning and leaf flattening, respectively. NPH3 can act in both phot1 and phot2 pathways, and synergistic interactions observed between pks2 and nph3 mutants suggest complementary roles of PKS2 and NPH3 during phototropin signaling. Finally, several observations further suggest that PKS2 may regulate leaf flattening and positioning by controlling auxin homeostasis. Together with previous findings, our results indicate that the PKS proteins represent an important family of phototropin signaling proteins.Plants constantly monitor the properties of light in their natural environment to optimize light capture for photosynthesis and growth (e.g. shade avoidance and phototropism) and to time important developmental transitions (e.g. germination and flowering; Neff et al., 2000; Briggs and Christie, 2002; Franklin and Whitelam, 2005). To do so, plants have a multitude of photoreceptors that allow them to sense changes in light period, direction, wavelength composition, and intensity. The main types of photoreceptors are the red/far-red light-absorbing phytochromes and the UV-A/blue light-sensing phototropins, cryptochromes, and Zeitlupe protein families (Chen et al., 2004; Jiao et al., 2007; Demarsy and Fankhauser, 2009). The signaling pathways triggered by these photoreceptors are integrated to fine-tune responses to ever-changing light environments (Casal, 2000; Franklin and Whitelam, 2004; Iino, 2006).In Arabidopsis (Arabidopsis thaliana), phototropin1 (phot1) and its paralog phot2 were discovered as primary photoreceptors for blue light-induced hypocotyl phototropism and for high light-induced chloroplast avoidance movements, respectively (Liscum and Briggs, 1995; Huala et al., 1997; Jarillo et al., 2001; Kagawa et al., 2001). Subsequent studies have shown that phototropins regulate a wide set of physiological and developmental responses, including chloroplast accumulation under low light, stomatal opening, leaf flattening, and phototropism of the root, inflorescence stem, and petiole (Sakai et al., 2001). Thus, phototropins are proposed to optimize the photosynthetic potential of plants, particularly under unfavorable environments such as extremely high light, weak illumination, and drought (Kasahara et al., 2002; Takemiya et al., 2005; Galen et al., 2007).Phot1 and phot2 regulate these processes selectively and in a fluence-dependent manner. Phot1 mediates the chloroplast accumulation, leaf positioning, and phototropic responses under very low light (Demarsy and Fankhauser, 2009). Under higher light intensities, the phot2 pathway becomes activated and acts redundantly with phot1 in these processes (Sakai et al., 2001). Phot2 also specifically controls the chloroplast avoidance response induced by high light (Jarillo et al., 2001; Kagawa et al., 2001). For stomatal opening, phot1 and phot2 act redundantly over a broad range of light intensity (Kinoshita et al., 2001; Doi et al., 2004).Phototropins are Ser/Thr kinases belonging to the AGC family (for cAMP-dependent protein kinase, cGMP-dependent protein kinase, and phospholipids-dependent protein kinase C; Bogre et al., 2003). Two LOV (for light, oxygen, or voltage) photosensory domains that bind to the blue light-absorbing chromophore FMN regulate the kinase activity (Christie, 2007). Phototropin activation and early signaling events at the level of the photoreceptor itself have been extensively studied (Tokutomi et al., 2008; Demarsy and Fankhauser, 2009). However, downstream signaling is less well understood. Light-induced phot1 autophosphorylation has recently been shown to be an essential signaling event, but apart from the photoreceptor itself, no direct substrate for the kinase activity has been identified in planta (Inoue et al., 2008b; Sullivan et al., 2008). Nonetheless, several proteins are known to interact with phot1. These include Broad-Complex, Tramtrack, Bric-à-Brac (BTB) proteins belonging to the 33-member NONPHOTOTROPIC HYPOCOTYL3 (NPH3)/ROOT PHOTOTROPISM2-LIKE (NRL) subfamily, 14-3-3 proteins, and ADP-ribosylation factors (members of the Ras superfamily of GTP-binding proteins that play important roles in the assembly and disassembly of coat proteins associated with driving vesicle budding and fusion; Motchoulski and Liscum, 1999; Sullivan et al., 2009).Genetic experiments showed that NPH3 is required for phot1- and phot2-mediated phototropism and for phot1-controlled leaf positioning but is not involved in stomatal opening or chloroplast movements (Inada et al., 2004; Inoue et al., 2008a). In addition, RPT2 acts in the phot1-induced phototropic response and stomatal opening but not in chloroplast relocation or phot2-induced movements. RPT2 can associate with phot1 in vitro and in vivo, but there is no evidence for a direct interaction with phot2 (Inada et al., 2004). NPH3 is also known to interact with phot1 in vivo, but an interaction with phot2 has not been reported (Motchoulski and Liscum, 1999; Lariguet et al., 2006). Thus, phototropin signaling is believed to branch quickly, and phot1 and phot2 appear to recruit different signaling components to trigger distinct physiological processes. NPH3 and RPT2 are proposed to mediate protein scaffolding using their protein-protein interaction domains (BTB/Pox virus and Zinc finger as well as coiled coil) and by these means may provide signaling specificity via interaction with specific targets in different tissues and subcellular compartments (Celaya and Liscum, 2005). The phototropins may regulate such interactions by modifying the phosphorylation status of the signaling protein (e.g. NPH3 and 14-3-3 proteins; Pedmale and Liscum, 2007; Sullivan et al., 2009).The nature of phototropin-controlled responses is diverse. On the one hand, chloroplast movements and stomatal opening are rapid, cell-autonomous, and reversible processes. On the other hand, phototropic responses and leaf flattening are slower symmetric growth processes coordinated by cell expansion and division. Such growth coordination is under tight hormonal regulation, and the hormone auxin is a central regulator of phototropism (Holland et al., 2009), leaf flattening (Keller and Van Volkenburgh, 1997; Li et al., 2007; Bainbridge et al., 2008; Braun et al., 2008), and leaf positioning (Tao et al., 2008; Millenaar et al., 2009). An important task is to identify points of convergence between phototropin signaling and auxin signaling. Hypocotyl phototropism is triggered by blue light-induced auxin redistribution and signaling across the organ (Esmon et al., 2006; Holland et al., 2009). Recent reports suggest that the phototropins achieve this by directly regulating the activity of auxin transporters. First, the three main classes of auxin transporters (AUXIN RESISTANT1 AUX1]/LIKE AUX1, PIN-FORMED PIN], and P-glycoproteins PGP]) are involved in the regulation of phototropism (Friml et al., 2002; Noh et al., 2003; Blakeslee et al., 2004; Nagashima et al., 2008; Stone et al., 2008). Second, phot1 is required for the relocalization of PIN1 upon blue light exposure (Blakeslee et al., 2004). Third, the phototropin-related AGC kinase PINOID (PID) is a crucial regulator of PIN1 intracellular cycling, which suggests an important role for AGC kinases in the regulation of auxin transport polarity (Michniewicz et al., 2007; Robert and Offringa, 2008). The link between the phototropins and auxin has not been firmly established in the cases of leaf flattening and leaf positioning.NPH3 is a strong candidate to provide a link between phototropins and auxin transport. First, NPH3 acts specifically in phototropin-controlled processes that involve growth regulation. Second, the rice (Oryza sativa) homolog of NPH3 called COLEOPTILE PHOTOTROPISM1 (CPT1) is an essential mediator of auxin redistribution in coleoptiles during the phototropin response (Haga et al., 2005). Third, an Arabidopsis homolog of NPH3 named MACCHIBOU4/ENHANCER OF PINOID/NAKED PINS IN YUC MUTANTS1 (MAB4/ENP/NPY1) is involved in organogenesis synergistically with PID by controlling PIN1 localization in embryo and inflorescence stems (Cheng et al., 2007; Furutani et al., 2007). However, beyond these correlative observations, the mechanisms of auxin transport regulation by phototropin signaling remain poorly understood (Holland et al., 2009).PHYTOCHROME KINASE SUBSTRATE (PKS) proteins were initially identified as phytochrome signaling components that regulate developmental processes such as deetiolation and growth orientation of roots and hypocotyls (Fankhauser et al., 1999; Lariguet et al., 2003; Khanna et al., 2006; Boccalandro et al., 2008; Molas and Kiss, 2008; Schepens et al., 2008). PKS1, PKS2, and PKS4 interact with phytochrome A and PKS1 is phosphorylated by phytochrome A in vitro (Fankhauser et al., 1999; Lariguet et al., 2003; Schepens et al., 2008). Recently, we have shown that PKS1 also interacts with phot1 and NPH3 in vivo and is required for phot1-mediated root and hypocotyl phototropism (Lariguet et al., 2006; Boccalandro et al., 2008). The importance of PKS proteins for phototropism prompted us to test their involvement in phototropin-mediated responses more globally. Here, we show that PKS2 acts in phot1 and phot2 signaling pathways controlling leaf positioning and leaf flattening but not chloroplast movements and stomatal opening. Interestingly, PKS2 and NPH3 selectively control phototropin-mediated growth responses and interact genetically. Several lines of evidence, including auxin transport assays in mesophyll protoplasts, suggest that PKS2 may regulate these developmental light responses by modulating auxin homeostasis. |
