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.     

RPT2 is a signal transducer involved in phototropic response and stomatal opening by association with phototropin 1 in Arabidopsis thaliana

Phototropin 1 (phot1) and phot2, which are blue light receptor kinases, function in blue light-induced hypocotyl phototropism, chloroplast relocation, and stomatal opening in Arabidopsis (Arabidopsis thaliana). Previous studies have shown that the proteins RPT2 (for ROOT PHOTOTROPISM2) and NPH3 (for NONPHOTOTROPIC HYPOCOTYL3) transduce signals downstream of phototropins to induce the phototropic response. However, the involvement of RPT2 and NPH3 in stomatal opening and in chloroplast relocation mediated by phot1 and phot2 was unknown. Genetic analysis of the rpt2 mutant and of a series of double mutants indicates that RPT2 is involved in the phot1-induced phototropic response and stomatal opening but not in chloroplast relocation or phot2-induced movements. Biochemical analyses indicate that RPT2 is purified in the crude microsomal fraction, as well as phot1 and NPH3, and that RPT2 makes a complex with phot1 in vivo. On the other hand, NPH3 is not necessary for stomatal opening or chloroplast relocation. Thus, these results suggest that phot1 and phot2 choose different signal transducers to induce three responses: phototropic response of hypocotyl, stomatal opening, and chloroplast relocation.     

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.     

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.     

FHY1 and FHL act together to mediate nuclear accumulation of the phytochrome A photoreceptor

The phytochrome family of red/far-red photoreceptors is involved in the regulation of a wide range of developmental responses in plants. The Arabidopsis genome contains five phytochromes (phyA-E), among which phyA and phyB play the most important roles. Phytochromes localize to the cytosol in the dark and accumulate in the nucleus under light conditions, inducing specific phytochrome-mediated responses. Light-regulated nuclear accumulation of the phytochrome photoreceptors is therefore considered a key regulatory step of these pathways. In fact, one of the most severe phyA signaling mutants, fhy1 (far red elongated hypocotyl 1), is strongly affected in nuclear accumulation of phyA. The fhy1 fhl (fhy1 like) double mutant, lacking both FHY1 and its only close homolog FHL, is virtually blind to far-red light like phyA null seedlings. Here we show that FHL accounts for residual amounts of phyA in the nucleus in a fhy1 background and that nuclear accumulation of phyA is completely inhibited in an fhy1 FHL RNAi knock-down line. Moreover, we demonstrate that FHL and phyA interact with each other in a light-dependent manner and that they co-localize in light-induced nuclear speckles. We also identify a phyA-binding site at the C-terminus of FHY1 and FHL, and show that the N-terminal 406 amino acids of phyA are sufficient for the interaction with FHY1/FHL.     

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.     

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.     

Phytochrome Kinase Substrate 4 is phosphorylated by the phototropin 1 photoreceptor

Phototropism allows plants to redirect their growth towards the light to optimize photosynthesis under reduced light conditions. Phototropin 1 (phot1) is the primary low blue light-sensing receptor triggering phototropism in Arabidopsis. Light-induced autophosphorylation of phot1, an AGC-class protein kinase, constitutes an essential step for phototropism. However, apart from the receptor itself, substrates of phot1 kinase activity are less clearly established. Phototropism is also influenced by the cryptochromes and phytochromes photoreceptors that do not provide directional information but influence the process through incompletely characterized mechanisms. Here, we show that Phytochrome Kinase Substrate 4 (PKS4), a known element of phot1 signalling, is a substrate of phot1 kinase activity in vitro that is phosphorylated in a phot1-dependent manner in vivo. PKS4 phosphorylation is transient and regulated by a type 2-protein phosphatase. Moreover, phytochromes repress the accumulation of the light-induced phosphorylated form of PKS4 showing a convergence of photoreceptor activity on this signalling element. Our physiological analyses suggest that PKS4 phosphorylation is not essential for phototropism but is part of a negative feedback mechanism.     

RPT2. A signal transducer of the phototropic response in Arabidopsis

The blue light receptor NPH1 (for nonphototropic hypocotyl) has been considered to be the only UV-A/blue light receptor that induces a phototropic response by the hypocotyl and root of Arabidopsis. By analysis of root phototropism (rpt) mutants, we show, however, the involvement of another blue light receptor as well as the existence of two separate signaling pathways working downstream of these receptors in the phototropic response. A newly isolated gene, RPT2, controls one of these pathways. The RPT2 gene is light inducible; encodes a novel protein with putative phosphorylation sites, a nuclear localization signal, a BTB/POZ domain, and a coiled-coil domain; and belongs to a large gene family that includes the recently isolated NPH3 gene. From genetic, physiological, and biochemical evidence, we propose a genetic model of the signaling pathways that induce the phototropic response in Arabidopsis.     

Phosphorylation of NONPHOTOTROPIC HYPOCOTYL3 affects photosensory adaptation during the phototropic response

Photosensory adaptation, which can be classified as sensor or effector adaptation, optimizes the light sensing of living organisms by tuning their sensitivity to changing light conditions. During the phototropic response in Arabidopsis (Arabidopsis thaliana), the light-dependent expression controls of blue-light (BL) photoreceptor phototropin 1 (phot1) and its modulator ROOT PHOTOTROPISM2 (RPT2) are known as the molecular mechanisms underlying sensor adaptation. However, little is known about effector adaption in plant phototropism. Here, we show that control of the phosphorylation status of NONPHOTOTROPIC HYPOCOTYL3 (NPH3) leads to effector adaptation in hypocotyl phototropism. We generated unphosphorable and phosphomimetic NPH3 proteins on seven phosphorylation sites in the etiolated seedlings of Arabidopsis. Unphosphorable NPH3 showed a shortening of its retention time in the cytosol and caused an inability to adapt to very low fluence rates of BL (∼10⁻⁵ µmol m⁻² s⁻¹) during the phototropic response. In contrast, the phosphomimetic NPH3 proteins had a lengthened retention time in the cytosol and could not enable the adaptation to BL at fluence rates of 10⁻³ µmol m⁻² s⁻¹ or more. Our results indicate that the activation level of phot1 and the corresponding phosphorylation level of NPH3 determine the dissociation rate and the reassociation rate of NPH3 on the plasma membrane, respectively. These mechanisms may moderately maintain the active state of phot1 signaling across a broad range of BL intensities and contribute to the photosensory adaptation of phot1 signaling during the phototropic response in hypocotyls.

The phosphorylation status of NONPHOTOTROPIC HYPOCOTYL3 pr     

Intramolecular co-action of two independent photosensory modules in the fern phytochrome 3

Fern phytochrome3/neochrome1 (phy3/neo1) is a chimeric photoreceptor composed of a phytochrome-chromophore binding domain and an almost full-length phototropin. phy3 thus contains two different light-sensing modules; a red/far-red light receptor phytochrome and a blue light receptor phototropin. phy3 induces both red light- and blue light-dependent phototropism in phototropin-deficient Arabidopsis thaliana (phot1 phot2) seedlings. The red-light response is dependent on the phytochrome module of phy3, and the blue-light response is dependent on the phototropin module. We recently showed that both the phototropin-sensing module and the phytochrome-sensing module mediate the blue light-dependent phototropic response. Particularly under low-light conditions, these two light-sensing modules cooperate to induce the blue light-dependent phototropic response. This intramolecular co-action of two independent light-sensing modules in phy3 enhances light sensitivity, and perhaps allowed ferns to adapt to the low-light canopy conditions present in angiosperm forests.     

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.     

Arabidopsis FHY3 defines a key phytochrome A signaling component directly interacting with its homologous partner FAR1

In Arabidopsis, phytochrome A (phyA) is the primary photoreceptor mediating various plant responses to far-red (FR) light. Here we show that phyA signaling involves a combinatorial action of downstream intermediates, which controls overlapping yet distinctive sets of FR responses. FHY3 is a prominent phyA signaling intermediate sharing structural similarity to FAR1, a previously identified phyA signaling component. The fhy3 and far1 mutants display similar yet distinctive defects in phyA signaling; however, overexpression of either FHY3 or FAR1 suppresses the mutant phenotype of both genes. Moreover, overexpression of partial fragments of FHY3 can cause a dominant-negative interference phenotype on phyA signaling that is stronger than those of the fhy3 or far1 null mutants. Further, we demonstrate that FHY3 and FAR1 are capable of homo- and hetero-interaction. Our data indicate that FHY3, together with FAR1, defines a key module in a signaling network underlying phyA-mediated FR light responses.     

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.     

Cryptochrome‐mediated hypocotyl phototropism was regulated antagonistically by gibberellic acid and sucrose in Arabidopsis

Both phototropins(phot1 and phot2) and cryptochromes(cry1 and cry2) were proven as the Arabidopsis thaliana blue light receptors. Phototropins predominately function in photomovement, and cryptochromes play a role in photomorphogenesis. Although cryptochromes have been proposed to serve as positive modulators of phototropic responses, the underlying mechanism remains unknown. Here, we report that depleting sucrose from the medium or adding gibberellic acids(GAs) can partially restore the defects in phototropic curvature of the phot1 phot2 double mutants under high-intensity blue light; this restoration does not occur in phot1 phot2 cry1 cry2 quadruple mutants and nph3(nonphototropic hypocotyl 3) mutants which were impaired phototropic response in sucrose-containing medium. These results indicate that GAs and sucrose antagonistically regulate hypocotyl phototropism in a cryptochromes dependent manner, but it showed a crosstalk with phototropin signaling on NPH3.Furthermore, cryptochromes activation by blue light inhibit GAs synthesis, thus stabilizing DELLAs to block hypocotyl growth, which result in the higher GAs content in the shade side than the lit side of hypocotyl to support the asymmetric growth of hypocotyl. Through modulation of the abundance of DELLAs by sucrose depletion or added GAs, it revealed that cryptochromes have a function in mediating phototropic curvature.     

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.