Reduced phototropism in pks mutants may be due to altered auxin‐regulated gene expression or reduced lateral auxin transport

Phototropism allows plants to orient their photosynthetic organs towards the light. In Arabidopsis, phototropins 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. Phototropism results from asymmetric growth in the hypocotyl elongation zone that depends on an auxin gradient 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 phototropism. 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.     

PKS1 plays a role in red-light-based positive phototropism in roots

Aerial parts of plants curve towards the light (i.e. positive phototropism), and roots typically grow away from the light (i.e. negative phototropism). In addition, Arabidopsis roots exhibit positive phototropism relative to red light (RL), and this response is mediated by phytochromes A and B (phyA and phyB). Upon light stimulation, phyA and phyB interact with the phytochrome kinase substrate (PKS1) in the cytoplasm. In this study, we investigated the role of PKS1, along with phyA and phyB, in the positive phototropic responses to RL in roots. Using a high-resolution feedback system, we studied the phenotypic responses of roots of phyA, phyB, pks1, phyA pks1 and phyB pks1 null mutants as well as the PKS1-overexpressing line in response to RL. PKS1 emerged as an intermediary in the signalling pathways and appears to promote a negative curvature to RL in roots. In addition, phyA and phyB were both essential for a positive response to RL and act in a complementary fashion. However, either photoreceptor acting without the other results in negative curvature in response to red illumination so that the mode of action differs depending on whether phyA and phyB act independently or together. Our results suggest that PKS1 is part of a signalling pathway independent of phyA and phyB and that PKS1 modulates RL-based root phototropism.     

Regulation of phototropic signaling in Arabidopsis via phosphorylation state changes in the phototropin 1-interacting protein NPH3

Phototropism, or the directional growth (curvature) of various organs toward or away from incident light, represents a ubiquitous adaptive response within the plant kingdom. This response is initiated through the sensing of directional blue light (BL) by a small family of photoreceptors known as the phototropins. Of the two phototropins present in the model plant Arabidopsis thaliana, phot1 (phototropin 1) is the dominant receptor controlling phototropism. Absorption of BL by the sensory portion of phot1 leads, as in other plant phototropins, to activation of a C-terminal serine/threonine protein kinase domain, which is tightly coupled with phototropic responsiveness. Of the five phot1-interacting proteins identified to date, only one, NPH3 (non-phototropic hypocotyl 3), is essential for all phot1-dependent phototropic responses, yet little is known about how phot1 signals through NPH3. Here, we show that, in dark-grown seedlings, NPH3 exists as a phosphorylated protein and that BL stimulates its dephosphorylation. phot1 is necessary for this response and appears to regulate the activity of a type 1 protein phosphatase that catalyzes the reaction. The abrogation of both BL-dependent dephosphorylation of NPH3 and development of phototropic curvatures by protein phosphatase inhibitors further suggests that this post-translational modification represents a crucial event in phot1-dependent phototropism. Given that NPH3 may represent a core component of a CUL3-based ubiquitin-protein ligase (E3), we hypothesize that the phosphorylation state of NPH3 determines the functional status of such an E3 and that differential regulation of this E3 is required for normal phototropic responsiveness.     

Hypocotyl growth orientation in blue light is determined by phytochrome A inhibition of gravitropism and phototropin promotion of phototropism

How developing seedlings integrate gravitropic and phototropic stimuli to determine their direction of growth is poorly understood. In this study we tested whether blue light influences hypocotyl gravitropism in Arabidopsis. Phototropin1 (phot1) triggers phototropism under low fluence rates of blue light but, at least in the dark, has no effect on gravitropism. By analyzing the growth orientation of phototropism-deficient seedlings in response to gravitropic and phototropic stimulations we show that blue light not only triggers phototropism but also represses hypocotyl gravitropism. At low fluence rates of blue light phot1 mutants were agravitropic. In contrast, phyAphot1 double mutants grew exclusively according to gravity demonstrating that phytochrome A (phyA) is necessary to inhibit gravitropism. Analyses of phot1cry1cry2 triple mutants indicate that cryptochromes play a minor role in this response. Thus the optimal growth orientation of hypocotyls is determined by the action of phyA-suppressing gravitropism and the phototropin-triggering phototropism. It has long been known that phytochromes promote phototropism but the mechanism involved is still unknown. Our data show that by inhibiting gravitropism phyA acts as a positive regulator of phototropism.     

Functional analyses of the activation loop of phototropin2 in Arabidopsis

Phototropins (phot1 and phot2) are autophosphorylating blue-light receptor kinases that mediate blue-light responses such as phototropism, chloroplast accumulation, and stomatal opening in Arabidopsis (Arabidopsis thaliana). Only phot2 induces the chloroplast avoidance response under strong blue light. The serine (Ser) residues of the kinase activation loop in phot1 are autophosphorylated by blue light, and autophosphorylation is essential for the phot1-mediated responses. However, the role of autophosphorylation in phot2 remains to be determined. In this study, we substituted the conserved residues of Ser-761 and Ser-763 with alanine (S761A S763A) in the phot2 activation loop and analyzed their function by investigating the phot2-mediated responses after the transformation of phot1 phot2 double mutant with this mutant phot2 gene. Transgenic plants expressing the mutant phot2 protein exhibited impaired responses in chloroplast movement, stomatal opening, phototropic bending, leaf flattening, and plant growth; and those expressing phot2 with S761D S763D mutations showed the normal responses. Substitution of both Ser-761 and Ser-763 with alanine in phot2 did not significantly affect the kinase activity in planta. From these results, we conclude that phosphorylation of Ser-761 and Ser-763 in the activation loop may be a common primary step for phot2-mediated responses.     

The Arabidopsis rcn1-1 Mutation Impairs Dephosphorylation of Phot2, Resulting in Enhanced Blue Light Responses

Phototropins (phot) sense blue light through the two N-terminal chromophore binding LOV domains and activate the C-terminal kinase domain. The resulting phototropin autophosphorylation is essential for biological activity. We identified the A1 subunit of Ser/Thr protein phosphatase 2A (PP2A) as interacting with full-length phot2 in yeast and also interacting with phot2 in an in vitro protein binding assay. Phenotypic characterizations of a phot1-5 rcn1-1 (for root curling in n-naphthylphthalamic acid1) double mutant, in which phot2 is the only functional phototropin and PP2A activity is reduced, showed enhanced phototropic sensitivity and enhanced blue light–induced stomatal opening, suggesting that PP2A activity is involved in regulating phot2 function. When treated with cantharidin, a chemical inhibitor of PP2A, the phot1-5 mutant exhibited enhanced phot2-mediated phototropic responses like those of the phot1-5 rcn1-1 double mutant. Immunoblot analysis to examine phot2 endogenous phosphorylation levels and in vitro phosphorylation assays of phot2 extracted from plants during dark recovery from blue light exposure confirmed that phot2 is more slowly dephosphorylated in the reduced PP2A activity background than in the wild-type PP2A background, suggesting that phosphorylated phot2 is a substrate of PP2A activity. While reduced PP2A activity enhanced the activity of phot2, it did not enhance either phot1 dephosphorylation or the activity of phot1 in mediating phototropism or stomatal opening.     

An experimental test of the adaptive evolution of phototropins: blue-light photoreceptors controlling phototropism in Arabidopsis thaliana

Phototropins are blue-light photoreceptor molecules mediating the capacity for phototropism or bending toward or away from directional light. Like the red-light sensing phytochromes that control shade avoidance, phototropins modulate developmental plasticity in plant architecture. Yet, unlike phytochromes, the adaptive significance of phototropins has been largely a topic of conjecture. In Arabidopsis thaliana, phototropism of seedling and plant stems is under the control of two paralogous genes, PHOT1 and PHOT2, that encode different phototropins with partially redundant light response qualities. The PHOT1 gene product interacts with the NPH3 gene product to cause phototropic bending over a broad range of light intensity, from very weak light in the soil to stronger light in the aerial environment. The PHOT2 gene product modulates shoot bending in response to light of higher intensity only. We compared the fitness of wild-type, phot1, phot2, and nph3 genotypes over a range of light conditions in the field. Seeds were sown in the field on the soil surface and left bare or covered with either gravel or bark mulch chips. Plantings were made under full sun and dense canopy cover. Rates of seedling emergence, survival to flowering, and total seed set were measured. All mutant genotypes had significantly reduced lifetime fitness compared to wild-type. Consistent with their different fluence rate sensitivities, phot1 and phot2 signaling pathways affected fitness at discrete life-cycle stages. Fitness costs of phot1 and nph3 were expressed mainly during seedling emergence from the soil whereas that of phot2 was expressed solely after emergence. Surprisingly, the only significant genotype-by-environment interaction for fitness occurred during emergence: genotypes blind to dim blue light (phot1 and nph3) had poor emergence in the open, but not in the shade. Possibly, the loss of negative phototropism in seedling roots of mutant genotypes reduced establishment success in open (dry soil) conditions. Results show that phototropin-modulated pathways are adaptive and that their evolution has involved functional specialization. However, mechanism(s) of selection on these pathways remain a mystery.     

Functional characterization of Arabidopsis phototropin 1 in the hypocotyl apex

Phototropin (phot1) is a blue light‐activated plasma membrane‐associated kinase that acts as the principal photoreceptor for shoot phototropism in Arabidopsis in conjunction with the signalling component Non‐Phototropic Hypocotyl 3 (NPH3). PHOT1 is uniformly expressed throughout the Arabidopsis hypocotyl, yet decapitation experiments have localized the site of light perception to the upper hypocotyl. This prompted us to investigate in more detail the functional role of the hypocotyl apex, and the regions surrounding it, in establishing phototropism. We used a non‐invasive approach where PHOT1–GFP (P1–GFP) expression was targeted to the hypocotyl apex of the phot‐deficient mutant using the promoters of CUP‐SHAPED COTYLEDON 3 (CUC3) and AINTEGUMENTA (ANT). Expression of CUC3::P1–GFP was clearly visible at the hypocotyl apex, with weaker expression in the cotyledons, whereas ANT::P1–GFP was specifically targeted to the developing leaves. Both lines showed impaired curvature to 0.005 μmol m^?2 sec^?1 unilateral blue light, indicating that regions below the apical meristem are necessary for phototropism. Curvature was however apparent at higher fluence rates. Moreover, CUC3::P1–GFP partially or fully complemented petiole positioning, leaf flattening and chloroplast accumulation, but not stomatal opening. Yet, tissue analysis of NPH3 de‐phosphorylation showed that CUC3::P1–GFP and ANT::P1–GFP mis‐express very low levels of phot1 that likely account for this responsiveness. Our spatial targeting approach therefore excludes the hypocotyl apex as the site for light perception for phototropism and shows that phot1‐mediated NPH3 de‐phosphorylation is tissue autonomous and occurs more prominently in the basal hypocotyl.     

Arabidopsis ROOT PHOTOTROPISM2 Contributes to the Adaptation to High-Intensity Light in Phototropic Responses

Living organisms adapt to changing light environments via mechanisms that enhance photosensitivity under darkness and attenuate photosensitivity under bright light conditions. In hypocotyl phototropism, phototropin1 (phot1) blue light photoreceptors mediate both the pulse light-induced, first positive phototropism and the continuous light-induced, second positive phototropism, suggesting the existence of a mechanism that alters their photosensitivity. Here, we show that light induction of ROOT PHOTOTROPISM2 (RPT2) underlies photosensory adaptation in hypocotyl phototropism of Arabidopsis thaliana. rpt2 loss-of-function mutants exhibited increased photosensitivity to very low fluence blue light but were insensitive to low fluence blue light. Expression of RPT2 prior to phototropic stimulation in etiolated seedlings reduced photosensitivity during first positive phototropism and accelerated second positive phototropism. Our microscopy and biochemical analyses indicated that blue light irradiation causes dephosphorylation of NONPHOTOTROPIC HYPOCOTYL3 (NPH3) proteins and mediates their release from the plasma membrane. These phenomena correlate closely with the desensitization of phot1 signaling during the transition period from first positive phototropism to second positive phototropism. RPT2 modulated the phosphorylation of NPH3 and promoted reconstruction of the phot1-NPH3 complex on the plasma membrane. We conclude that photosensitivity is increased in the absence of RPT2 and that this results in the desensitization of phot1. Light-mediated induction of RPT2 then reduces the photosensitivity of phot1, which is required for second positive phototropism under bright light conditions.     

The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 Protein Is a Phototropin Signaling Element That Regulates Leaf Flattening and Leaf Positioning

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.     

Physiological roles of the light, oxygen, or voltage domains of phototropin 1 and phototropin 2 in Arabidopsis

Phototropins (phot1 and phot2) are plant blue-light receptors that mediate phototropism, chloroplast movement, stomatal opening, rapid inhibition of growth of etiolated seedlings, and leaf expansion in Arabidopsis (Arabidopsis thaliana). Their N-terminal region contains two light, oxygen, or voltage (LOV) domains, which bind flavin mononucleotide and form a covalent adduct between a conserved cysteine and the flavin mononucleotide chromophore upon photoexcitation. The C-terminal region contains a serine/threonine kinase domain that catalyzes blue-light-activated autophosphorylation. Here, we have transformed the phot1 phot2 (phot1-5 phot2-1) double mutant with PHOT expression constructs driven by the cauliflower mosaic virus 35S promoter. These constructs encode either wild-type phototropin or phototropin with one or both LOV-domain cysteines mutated to block their photochemistry. We selected multiple lines in each of the eight resulting categories of transformants for further physiological analyses. Specifically, we investigated whether LOV1 and LOV2 serve the same or different functions for phototropism and leaf expansion. Our results show that the LOV2 domain of phot1 plays a major role in phototropism and leaf expansion, as does the LOV2 domain of phot2. No complementation of phototropism or leaf expansion was observed for the LOV1 domain of phot1. However, phot2 LOV1 was unexpectedly found to complement phototropism to a considerable level. Similarly, transformants carrying a PHOT transgene with both LOV domains inactivated developed strong curvatures toward high fluence rate blue light. However, we found that the phot2-1 mutant is leaky and produces a small level of full-length phot2 protein. In vitro experiments indicate that cross phosphorylation can occur between functional phot2 and inactivated phot1 molecules. Such a mechanism may occur in vivo and therefore account for the functional activities observed in the PHOT transgenics with both lov domains inactivated. The implications of this mechanism with respect to phototropin function are discussed.     

Subcellular localization and turnover of Arabidopsis phototropin 1

We reported recently that internalization of the plant blue light receptor phototropin 1 (phot1) from the plasma membrane in response to irradiation is reliant on receptor autophosphorylation. Pharmacological interference and co-immunoprecipitation analyses also indicated that light-induced internalization of phot1 involves clathrin-dependent processes. Here, we describe additional pharmacological studies that impact the subcellular localization and trafficking of Arabidopsis phot1. Alterations in the microtububle cytoskeleton led to dramatic differences in phot1 localization and function. Likewise, inhibition of phosphatidic acid (PA) signaling was found to impair phot1 localization and function. However, action of PA inhibition on phot1 function may be attributed to pleiotropic effects on cell growth. While phot1 kinase activation is necessary to stimulate its internalization, autophosphorylation is not required for phot1 turnover in response to prolonged blue light irradiation. The implications of these findings in regard to phot1 localization and function are discussed.Key words: phototropin 1 (phot1), phototropism, subcellular trafficking, autophosphorylation, protein turnover     

FERONIA is involved in phototropin 1-mediated blue light phototropic growth in Arabidopsis

Plant shoot phototropism is triggered by the formation of a light-driven auxin gradient leading to bending growth. The blue light receptor phototropin 1 (phot1) senses light direction, but how this leads to auxin gradient formation and growth regulation remains poorly understood. Previous studies have suggested phot1's role for regulated apoplastic acidification, but its relation to phototropin and hypocotyl phototropism is unclear. Herein, we show that blue light can cause phot1 to interact with and phosphorylate FERONIA (FER), a known cell growth regulator, and trigger downstream phototropic bending growth in Arabidopsis hypocotyls. fer mutants showed defects in phototropic growth, similar to phot1/2 mutant. FER also interacts with and phosphorylates phytochrome kinase substrates, the phot1 downstream substrates. The phot1-FER pathway acts upstream of apoplastic acidification and the auxin gradient formation in hypocotyl under lateral blue light, both of which are critical for phototropic bending growth in hypocotyls. Our study highlights a pivotal role of FER in the phot1-mediated phototropic cell growth regulation in plants.     

PHYTOCHROME KINASE SUBSTRATE4 modulates phytochrome-mediated control of hypocotyl growth orientation

Gravity and light are major factors shaping plant growth. Light perceived by phytochromes leads to seedling deetiolation, which includes the deviation from vertical hypocotyl growth and promotes hypocotyl phototropism. These light responses enhance survival of young seedlings during their emergence from the soil. The PHYTOCHROME KINASE SUBSTRATE (PKS) family is composed of four members in Arabidopsis (Arabidopsis thaliana): PKS1 to PKS4. Here we show that PKS4 is a negative regulator of both phytochrome A- and B-mediated inhibition of hypocotyl growth and promotion of cotyledon unfolding. Most prominently, pks4 mutants show abnormal phytochrome-modulated hypocotyl growth orientation. In dark-grown seedlings hypocotyls change from the original orientation defined by seed position to the upright orientation defined by gravity and light reduces the magnitude of this shift. In older seedlings with the hypocotyls already oriented by gravity, light promotes the deviation from vertical orientation. Based on the characterization of pks4 mutants we propose that PKS4 inhibits changes in growth orientation under red or far-red light. Our data suggest that in these light conditions PKS4 acts as an inhibitor of asymmetric growth. This hypothesis is supported by the phenotype of PKS4 overexpressers. Together with previous findings, these results indicate that the PKS family plays important functions during light-regulated tropic growth responses.     

PHYTOCHROME KINASE SUBSTRATE1 regulates root phototropism and gravitropism

Light promotes the expression of PHYTOCHROME KINASE SUBSTRATE1 (PKS1) in the root of Arabidopsis thaliana, but the function of PKS1 in this organ is unknown. Unilateral blue light induced a negative root phototropic response mediated by phototropin 1 in wild-type seedlings. This response was absent in pks1 mutants. In the wild type, unilateral blue light enhanced PKS1 expression in the subapical region of the root several hours before bending was detectable. The negative phototropism and the enhanced PKS1 expression in response to blue light required phytochrome A (phyA). In addition, the pks1 mutation enhanced the root gravitropic response when vertically oriented seedlings were placed horizontally. The negative regulation of gravitropism by PKS1 occurred even in dark-grown seedlings and did not require phyA. Blue light also failed to induce negative phototropism in pks1 under reduced gravitational stimulation, indicating that the effect of pks1 on phototropism is not simply the consequence of the counteracting effect of enhanced gravitropism. We propose a model where the background level of PKS1 reduces gravitropism. After a phyA-dependent increase in its expression, PKS1 positively affects root phototropism and both effects contribute to negative curvature in response to unilateral blue light.     

Phototropins Function in High-Intensity Blue Light-Induced Hypocotyl Phototropism in Arabidopsis by Altering Cytosolic Calcium

Phototropins (phot1 and phot2), the blue light receptors in plants, regulate hypocotyl phototropism in a fluence-dependent manner. Especially under high fluence rates of blue light (HBL), the redundant function mediated by both phot1 and phot2 drastically restricts the understanding of the roles of phot2. Here, systematic analysis of phototropin-related mutants and overexpression transgenic lines revealed that HBL specifically induced a transient increase in cytosolic Ca²⁺ concentration ([Ca2+]_cyt) in Arabidopsis (Arabidopsis thaliana) hypocotyls and that the increase in [Ca2+]_cyt was primarily attributed to phot2. Pharmacological and genetic experiments illustrated that HBL-induced Ca²⁺ increases were modulated differently by phot1 and phot2. Phot2 mediated the HBL-induced increase in [Ca2+]_cyt mainly by an inner store-dependent Ca²⁺-release pathway, not by activating plasma membrane Ca²⁺ channels. Further analysis showed that the increase in [Ca2+]_cyt was possibly responsible for HBL-induced hypocotyl phototropism. An inhibitor of auxin efflux carrier exhibited significant inhibitions of both phototropism and increases in [Ca2+]_cyt, which indicates that polar auxin transport is possibly involved in HBL-induced responses. Moreover, PHYTOCHROME KINASE SUBSTRATE1 (PKS1), the phototropin-related signaling element identified, interacted physically with phototropins, auxin efflux carrier PIN-FORMED1 and calcium-binding protein CALMODULIN4, in vitro and in vivo, respectively, and HBL-induced phototropism was impaired in pks multiple mutants, indicating the role of the PKS family in HBL-induced phototropism. Together, these results provide new insights into the functions of phototropins and highlight a potential integration point through which Ca²⁺ signaling-related HBL modulates hypocotyl phototropic responses.Blue light (BL) is a key factor controlling plant growth and morphogenesis. Recent genetics investigations using Arabidopsis (Arabidopsis thaliana) have revealed that the BL receptors phototropin1 (phot1) and phot2 mediate BL-induced plant movements such as phototropism, chloroplast relocation, stomatal opening, leaf flattening, and leaf positioning responses (Inoue et al., 2010). Most of these responses are mediated redundantly by both phot1 and phot2 (Kinoshita et al., 2001; Sakamoto and Briggs, 2002), but some responses are mediated by either phot1 or phot2 (Sakai et al., 2001; Suetsugu et al., 2005). In addition, several lines of evidence have indicated that phot2 might negatively regulate the phot1-mediated response (de Carbonnel et al., 2010) and vice versa (Harada et al., 2003, 2013).One of the numerous physiological processes controlled by BL is phototropism. Phototropism enables plants to bend toward incident light by perceiving the direction, wavelength, and intensity of incident light so that they are able to obtain optimum light. Genetic evidence has shown that both phot1 and phot2 redundantly function to regulate hypocotyl phototropism in a fluence-dependent manner (Sakai et al., 2001). Phot1 functions at both low (0.01–1 μmol m⁻² s⁻¹) and high (greater than 1 μmol m⁻² s⁻¹) fluence rates to mediate phototropic responses, but phot2 functions only at high fluence rates (Inada et al., 2004). The functional specification of phot1 and phot2 could be attributed to the differences in signal intermediates between phot1 and phot2 signaling pathways.Genetic analysis has illustrated that phot1 mediates hypocotyl phototropism via its downstream signal transducers NONPHOTOTROPIC HYPOCOTYL3 (NPH3; Motchoulski and Liscum, 1999), ROOT PHOTOTROPISM2 (RPT2; Sakai et al., 2000), and NONPHOTOTROPIC HYPOCOTYL4/AUXIN RESPONSE FACTOR7 (NPH4/ARF7; Harper et al., 2000), resulting in the asymmetric distribution of auxin and the induction of a phototropic response in higher plants. Recently, studies have demonstrated that PHYTOCHROME KINASE SUBSTRATE (PKS) proteins are required for hypocotyl phototropism and that PKS1 binds PHOT1 and NPH3 in vivo (Lariguet et al., 2006). In addition, ATP-BINDING CASSETTE B19 (ABCB19), a newly identified auxin transporter, has been reported to interact with phot1 to regulate the BL-dependent phototropism (Christie et al., 2011). However, little is known about phot2-mediated phototropism for functional specialization, especially under high fluence rates of blue light (HBL), although several lines of evidence have shown that phot2- and phot1-mediated signaling pathways share some intermediates in BL responses (Kimura and Kagawa, 2006; Christie, 2007). Previous researches have suggested that phot1 acts not only positively in the presence of RPT2 but also negatively in its absence during the phototropic response of hypocotyls at high fluence rates, suggesting that RPT2 modulates the function of phot1. However, RPT2 does not act in the phot2-mediated pathway (Inada et al., 2004). More recently, RCN1-1, the A1 subunit of Ser/Thr PROTEIN PHOSPHATASE2A (PP2A), has been identified to interact with phot2. While reduced PP2A activity enhances the activity of phot2, it does not enhance either phot1 dephosphorylation or the activity of phot1 in mediating phototropism (Tseng and Briggs, 2010).Besides these signal intermediates noted above, phototropins may also confer their effects through the change of ion homeostasis. Ca²⁺ is a case in point. Recent reports have demonstrated that phototropins mediate the mobilization of Ca²⁺ in response to BL and that phot1 and phot2 mediate Ca²⁺ increases with distinctive mechanism in leaf cells according to the changes of ambient light intensity (Harada and Shimazaki, 2007). Under low fluence rates of BL, phot1 solely mediated Ca²⁺ influx through the channels in the plasma membrane. Under HBL, the increase in cytosolic Ca²⁺ concentration ([Ca2+]_cyt) is primarily attributed to phot2-dependent Ca²⁺ release from the internal calcium stores as well as the plasma membrane Ca²⁺ channels. Interestingly, the inhibitory effects of phospholipase C (PLC) inhibitors on the BL-induced responses in the wild type are larger than those in the phot1 single mutant, which indicates that there are some functional interactions between phot1 and phot2 to induce the elevation of cytosolic Ca²⁺ (Harada et al., 2003).However, until now, the function of Ca²⁺ in the phototropin-mediated phototropism signaling process has remained largely unknown. Pharmacological experiments indicate that changes in [Ca2+]_cyt are required for the phot1-mediated inhibition of hypocotyl growth but not for phot1-mediated phototropism (Folta et al., 2003). Otherwise, electrophysiological studies indicate that phototropic bending involves changes in ion fluxes, including calcium (Babourina et al., 2004). Such divergent responses show that the link between phototropins and calcium has not been firmly established in the case of hypocotyl phototropism. In phototropism, the phot1-dependent relocalization of the auxin efflux carrier PIN-FORMED1 (PIN1) is required for auxin redistribution (Blakeslee et al., 2004), and the PINOID kinase influences the relocalization of PIN1 (Friml et al., 2004). Given that both the calmodulin-related protein TCH3 and the calcium-binding protein AtPBP1 can bind to the PINOID kinase (Benjamins et al., 2003), it would appear that the cross talk among phototropins, auxin, and calcium is an important event for phototropism.Here, we show that HBL induces increases in [Ca2+]_cyt, which are mostly attributed to the function of phot2, and that the increases in [Ca2+]_cyt are required for HBL-induced phototropism in Arabidopsis hypocotyls. We also demonstrate that PKS1 may integrate phototropins with auxin transport in phot2-dependent Ca²⁺ signaling, and we discuss the possible molecular link between phototropins and other potential signal elements in HBL-induced phototropism.     

Molecular basis of the functional specificities of phototropin 1 and 2

A blue-light photoreceptor in plants, phototropin, mediates phototropism, chloroplast relocation, stomatal opening, and leaf-flattening responses. Phototropin is divided into two functional moieties, the N-terminal photosensory and the C-terminal signaling moieties. Phototropin perceives light stimuli by the light, oxygen or voltage (LOV) domain in the N-terminus; the signal is then transduced intramolecularly to the C-terminal kinase domain. Two phototropins, phot1 and phot2, which have overlapping and distinct functions, exist in Arabidopsis thaliana. Phot1 mediates responses with higher sensitivity than phot2. Phot2 mediates specific responses, such as the chloroplast avoidance response and chloroplast dark positioning. To elucidate the molecular basis for the functional specificities of phot1 and phot2, we exchanged the N- and C-terminal moieties of phot1 and phot2, fused them to GFP and expressed them under the PHOT2 promoter in the phot1 phot2 mutant background. With respect to phototropism and other responses, the chimeric phototropin consisting of phot1 N-terminal and phot2 C-terminal moieties (P1n/2cG) was almost as sensitive as phot1; whereas the reverse combination (P2n/1cG) functioned with lower sensitivity. Hence, the N-terminal moiety mainly determined the sensitivity of the phototropins. Unexpectedly, both P1n/2cG and P2n/1cG mediated the chloroplast avoidance response, which is specific to phot2. Hence, chloroplast avoidance activity appeared to be suppressed specifically in the combination of N- and C-terminal moieties of phot1. Unlike the chloroplast avoidance response, chloroplast dark positioning was observed for P2G and P2n/1cG but not for P1G or P1n/2cG, suggesting that a specific structure in the N-terminal moiety of phot2 is required for this activity.     

植物蓝光受体研究进展

植物蓝光调节的反应主要有向光性、抑制幼茎伸长、叶绿体迁移、刺激气孔张开和调节基因表达等。蓝光反应的有效波长是蓝光和近紫外光(320—400am),故蓝光受体也叫蓝光／近紫外光受体。植物蓝光受体研究近年来取得较大进展。以拟南芥为例,已得到确认的受体至少有隐花色素(CRY1、2)和向光蛋白(phototropin)两大类。转基因拟南芥对蓝光、紫外光和绿光敏感,并发现CRY1是一个可溶性蛋白。CRY2编码一个核蛋白,蓝光在转录水平对该蛋白进行调节,它的作用是增加拟南芥对蓝光的敏感性。CRY1和CRY2共同介导了拟南芥植物的向光性。隐花色素的蛋白与辅基之间以非共价键连接,可以吸收蓝光和近紫外光。CRY1和CRY2蛋白之间,尤其是N端相似性很高。向光蛋白目前只发现PHOT1和PHO12两种,向光蛋白作为丝／苏氨酸激酶蓝光受体含有两个光氧化结构域(LOV)并参与了植物向光性叶绿体运动、气孔开放等。     

Phytochrome modulation of blue-light-induced phototropism

Red light enhances hypocotyl phototropism toward unilateral blue light through a phytochrome‐mediated response. This study demonstrates how the phytochromes modulate blue‐light‐induced phototropism in the absence of a red light pre‐treatment. It was found that phytochromes A, B, and D have conditionally overlapping functions in the promotion of blue‐light‐induced phototropism. Under very low blue light intensities (0.01 µmol m^?2 s^?1) phyA activity is necessary for the progression of a normal phototropic response, whereas above 1.0 µmol m^?1 s^?2 phyB and phyD have functional redundancy with phyA to promote phototropism. PhyA also contributes to attenuation of phototropism under high fluence rates of unilateral blue light, which was previously shown to be dependent on the phototropins and cryptochromes. From these results, it appears that phytochromes are required to develop a robust phototropic response under low fluence rates, whereas under high irradiances where phototropism may be less important, phyA suppresses phototropism.