Phototropin Mediated Relocation of Myosins in Arabidopsis thaliana

Background

The mechanism of the light-dependent movements of chloroplasts is based on actin and myosin but its details are largely unknown. The movements are activated by blue light in terrestrial angiosperms. The aim of the present study was to determine the role of myosin associated with the chloroplast surface in the light-induced chloroplast responses in Arabidopsis thaliana. The localization of myosins was investigated under blue light intensities generating avoidance and accumulation responses of chloroplasts. The localization was compared in wild type plants and in phot2 mutant lacking the avoidance response.

Results

Wild type and phot2 mutant plants were irradiated with strong (36 µEm⁻²s⁻¹) and/or weak (0.8 µEm⁻²s⁻¹) blue light. The leaf tissue was immunolabeled with antimyosin antibodies. Different arrangements of myosins were observed in the mesophyll depending on the fluence rate in wild type plants. In tissue irradiated with weak blue light myosins were associated with chloroplast envelopes. In contrast, in tissue irradiated with strong blue light chloroplasts were almost myosin-free. The effect did not occur in red light and in the phot2 mutant.

Conclusions

Myosin displacement is blue light specific, i.e., it is associated with the activation of a specific blue-light photoreceptor. We suggest that the reorganization of myosins is essential for chloroplast movement. Myosins appear to be the final step of the signal transduction pathway starting with phototropin2 and leading to chloroplast movements.Key Words: Arabidopsis, blue light, chloroplast movements, myosins, phototropins     

The phototropin family as photoreceptors for blue light-induced chloroplast relocation

Blue light-induced chloroplast accumulation and avoidance relocation movements are controlled by the blue light photoreceptor phototropin. The Arabidopsis thaliana genome has two phototropin genes encoding phot1 and phot2. Each of these photoreceptors contains two LOV (light oxygen and voltage) domains and a kinase domain. The LOV domains absorb blue light though an associated flavin mononucleotide chromophore, while the kinase domain is thought to be associated with signal transduction. The phototropins control not only chloroplast relocation movement, but also blue light-induced phototropic responses, leaf expansion and stomatal opening. Here I review the role of phototropin as a photoreceptor for chloroplast photorelocation movement. Electronic Publication     

Integrated role of ROS and Ca<Superscript>+2</Superscript> in blue light-induced chloroplast avoidance movement in leaves of <Emphasis Type="Italic">Hydrilla verticillata</Emphasis> (L.f.) Royle

Directional chloroplast photorelocation is a major physio-biochemical mechanism that allows these organelles to realign themselves intracellularly in response to the intensity of the incident light as an adaptive response. Signaling processes involved in blue light (BL)-dependent chloroplast movements were investigated in Hydrilla verticillata (L.f.) Royle leaves. Treatments with antagonists of actin filaments [2,3,5-triiodobenzoic acid (TIBA)] and microtubules (oryzalin) revealed that actin filaments, but not microtubules, play a pivotal role in chloroplast movement. Involvement of reactive oxygen species (ROS) in controlling chloroplast avoidance movement has been demonstrated, as exogenous H₂O₂ not only accelerated chloroplast avoidance but also could induce chloroplast avoidance even in weak blue light (WBL). Further support came from experiments with different ROS scavengers, i.e., dimethylthiourea (DMTU), KI, and CuCl₂, which inhibited chloroplast avoidance, and from ROS localization using specific stains. Such avoidance was also partially inhibited by ZnCl₂, an inhibitor of NADPH oxidase (NOX) as well as 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a photosynthetic electron transport chain (ETC) inhibitor at PS II. However, methyl viologen (MV), a PS I ETC inhibitor, rather accelerated avoidance response. Exogenous calcium (Ca⁺²) induced avoidance even in WBL while inhibited chloroplast accumulation partially. On the other hand, chloroplast movements (both accumulation and avoidance) were blocked by Ca⁺² antagonists, La³⁺ (inhibitor of plasma membrane Ca⁺² channel) and ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, Ca⁺² chelator) while LiCl that affects Ca⁺² release from endosomal compartments did not show any effect. A model on integrated role of ROS and Ca⁺² (influx from apolastic space) in actin-mediated chloroplast avoidance has been proposed.     

A Protein Phosphatase 2A Catalytic Subunit Modulates Blue Light-Induced Chloroplast Avoidance Movements through Regulating Actin Cytoskeleton in Arabidopsis

Chloroplast avoidance movements mediated by phototropin 2 (phot2) are one of most important physiological events in the response to high-fluence blue light (BL), which reduces damage to the photosynthetic machinery under excess light. Protein phosphatase 2A-2 (PP2A-2) is an isoform of the catalytic subunit of PP2A, which regulates a number of developmental processes. To investigate whether PP2A-2 was involved in high-fluence BL-induced chloroplast avoidance movements, we first analyzed chloroplast migration in the leaves of the pp2a-2 mutant in response to BL. The data showed that PP2A-2 might act as a positive regulator in phot2-mediated chloroplast avoidance movements, but not in phot1-mediated chloroplast accumulation movements. Then, the effect of okadaic acid (OA) and cantharidin (selective PP2A inhibitors) on high-fluence BL response was further investigated in Arabidopsis thaliana mesophyll cells. Within a certain concentration range, exogenously applied OA or cantharidin inhibited the high-fluence BL-induced chloroplast movements in a concentration-dependent manner. Actin depolymerizing factor (ADF)/cofilin phosphorylation assays demonstrated that PP2A-2 can activate/dephosphorylate ADF/cofilin, an actin-binding protein, in Arabidopsis mesophyll cells. Consistent with this observation, the experiments showed that OA could inhibit ADF1 binding to the actin and suppress the reorganization of the actin cytoskeleton after high-fluence BL irradiation. The adf1 and adf3 mutants also exhibited reduced high-fluence BL-induced chloroplast avoidance movements. In conclusion, we identified that PP2A-2 regulated the activation of ADF/cofilin, which, in turn, regulated actin cytoskeleton remodeling and was involved in phot2-mediated chloroplast avoidance movements.     

The role of a 14-3-3 protein in stomatal opening mediated by PHOT2 in Arabidopsis

The 14-3-3 λ isoform is required for normal stomatal opening mediated by PHOT2 in Arabidopsis thaliana. Arabidopsis phototropin2 (PHOT2) interacts with the λ-isoform 14-3-3 protein both in yeast two-hybrid screening and in an in vitro pull-down assay. Further yeast two-hybrid analysis also showed that the PHOT2 C-terminal kinase domain was required for the interaction. Site-directed mutagenesis indicated that PHOT2 Ser-747 is essential for the yeast interaction. Phenotypic characterization of a loss-of-function 14-3-3 λ mutant in a phot1 mutant background showed that the 14-3-3 λ protein was necessary for normal PHOT2-mediated blue light-induced stomatal opening. PHOT2 Ser-747 was necessary for complementation of the blue light-activated stomatal response in a phot1 phot2 double mutant. The 14-3-3 λ mutant in the phot1 mutant background allowed normal phototropism and normal chloroplast accumulation and avoidance responses. It also showed normal stomatal opening mediated by PHOT1 in a phot2 mutant background. The 14-3-3 κ mutant had no effect on stomatal opening in response to blue light. Although the 14-3-3 λ mutant had no chloroplast movement phenotype, the 14-3-3 κ mutation caused a weaker avoidance response at an intermediate blue light intensity by altering the balance between the avoidance and accumulation responses. The results highlight the strict specificity of phototropin-mediated signal transduction pathways.     

Blue light-induced chloroplast relocation

Chloroplast relocation movement is induced by blue light in most plants tested. Under weak light, chloroplasts move toward a brighter area in a cell (called low-fluence-rate response or accumulation movement), but they avoid strong light and move away from the light (called high-fluence-rate response or avoidance movement). Recently, mutants deficient in the chloroplast avoidance movement were isolated from Arabidopsis thaliana. The results of mutant analyses revealed that the phototropin photoreceptors phot1 and phot2 both control chloroplast accumulation while phot2 alone controls the avoidance movements.     

Red light, Phot1 and JAC1 modulate Phot2-dependent reorganization of chloroplast actin filaments and chloroplast avoidance movement

The phototropin (phot)-dependent intracellular relocation of chloroplasts is a ubiquitous phenomenon in plants. We have previously revealed the involvement of a short cp-actin (chloroplast actin) filament-based mechanism in this movement. Here, the reorganization of cp-actin filaments during the avoidance movement of chloroplasts was analyzed in higher time resolution under blue GFP (green fluorescent protein) excitation light in an actin filament-visualized line of Arabidopsis thaliana. Under standard background red light of 89 μmol m(-2) s(-1), cp-actin filaments transiently disappeared at approximately 30 s and reappeared in a biased configuration on chloroplasts approximately 70 s after blue excitation light irradiation. The timing of biased cp-actin reappearance was delayed under the background of strong red light or in the absence of red light. Consistently, chloroplast movement was delayed under these conditions. In phot1 mutants, acceleration of both the disappearance and reappearance of cp-actin filaments occurred, indicating an inhibitory action of phot1 on reorganization of cp-actin filaments. Avoidance movements began sooner in phot1 than in wild-type plants. No reorganization of cp-actin filaments was seen in phot2 or phot1phot2 mutants lacking phot2, which is responsible for avoidance movements. Surprisingly, jac1 (j-domain protein required for chloroplast accumulation response 1) mutants, lacking the accumulation response, showed no avoidance movements under the whole-cell irradiation condition for GFP observation. Cp-actin filaments in jac1 did not show a biased distribution, with a small or almost no transient decrease in the number. These results indicate a close association between the biased distribution of cp-actin filaments and chloroplast movement. Further, JAC1 is suggested to function in the biased cp-actin filament distribution by regulating their appearance and disappearance.     

Rapid Severing and Motility of Chloroplast-Actin Filaments Are Required for the Chloroplast Avoidance Response in Arabidopsis

Phototropins (phot1 and phot2 in Arabidopsis thaliana) relay blue light intensity information to the chloroplasts, which move toward weak light (the accumulation response) and away from strong light (the avoidance response). Chloroplast-actin (cp-actin) filaments are vital for mediating these chloroplast photorelocation movements. In this report, we examine in detail the cp-actin filament dynamics by which the chloroplast avoidance response is regulated. Although stochastic dynamics of cortical actin fragments are observed on the chloroplasts, the basic mechanisms underlying the disappearance (including severing and turnover) of the cp-actin filaments are regulated differently from those of cortical actin filaments. phot2 plays a pivotal role in the strong blue light–induced severing and random motility of cp-actin filaments, processes that are therefore essential for asymmetric cp-actin formation for the avoidance response. In addition, phot2 functions in the bundling of cp-actin filaments that is induced by dark incubation. By contrast, the function of phot1 is dispensable for these responses. Our findings suggest that phot2 is the primary photoreceptor involved in the rapid reorganization of cp-actin filaments that allows chloroplasts to change direction rapidly and control the velocity of the avoidance movement according to the light’s intensity and position.     

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.     

Phototropins promote plant growth in response to blue light in low light environments

Phototropins (phot1 and phot2) are plant-specific blue light receptors for phototropism, chloroplast movement, leaf expansion, and stomatal opening. All these responses are thought to optimize photosynthesis by helping to capture light energy efficiently, reduce photodamage, and acquire CO2. However, experimental evidence for the promotion of plant growth through phototropins is lacking. Here, we report dramatic phototropin-dependent effects on plant growth. When plants of Arabidopsis thaliana wild type, the phot1 and phot2 mutants, and the phot1 phot2 double mutant were grown under red light, no significant growth differences were observed. However, if a very low intensity of blue light (0.1 micromol m(-2) s(-1)) was superimposed on red light, large increases in fresh weight up to threefold were found in those plants that carried functional PHOT1 genes. When the intensity of blue light was increased to 1 micromol m(-2) s(-1), the growth enhancement was also found in the phot1 single mutant, but not in the double mutant, indicating that phot2 mediated similar responses as phot1 with a lower sensitivity. The effects occurred under low photosynthetically active radiation in particular. The well-known physiological phototropin-mediated responses, including chloroplast movement, stomatal opening, and leaf expansion, in the different lines tested indicated an involvement of these responses in the blue light-induced growth enhancement. We conclude that phototropins promote plant growth by controlling and integrating a variety of responses that optimize photosynthetic performance under low photosynthetically active radiation in the natural environment.     

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.     

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.     

Interaction between avoidance of photon absorption,excess energy dissipation and zeaxanthin synthesis against photooxidative stress in Arabidopsis

Plants evolved photoprotective mechanisms in order to counteract the damaging effects of excess light in oxygenic environments. Among them, chloroplast avoidance and non‐photochemical quenching concur in reducing the concentration of chlorophyll excited states in the photosynthetic apparatus to avoid photooxidation. We evaluated their relative importance in regulating excitation pressure on photosystem II. To this aim, genotypes were constructed carrying mutations impairing the chloroplast avoidance response (phot2) as well as mutations affecting the biosynthesis of the photoprotective xanthophyll zeaxanthin (npq1) or the activation of non‐photochemical quenching (npq4), followed by evaluation of their photosensitivity in vivo. Suppression of avoidance response resulted in oxidative stress under excess light at low temperature, while removing either zeaxanthin or PsbS had a milder effect. The double mutants phot2 npq1 and phot2 npq4 showed the highest sensitivity to photooxidative stress, indicating that xanthophyll cycle and qE have additive effects over the avoidance response. The interactions between non‐photochemical quenching and avoidance responses were studied by analyzing the kinetics of fluorescence decay and recovery at different light intensities. phot2 fluorescence decay lacked a component, here named as qM. This kinetic component linearly correlated with the leaf transmittance changes due to chloroplast relocation induced by white light and was absent when red light was used as actinic source. On these basis we conclude that a decrease in leaf optical density affects the apparent non‐photochemical quenching (NPQ) rise kinetic. Thus, excess light‐induced fluorescence decrease is in part due to avoidance of photon absorption rather than to a genuine quenching process.     

Actin cytoskeleton in Arabidopsis thaliana under blue and red light

BACKGROUND INFORMATION: Actin cytoskeleton is the basis of chloroplast-orientation movements. These movements are activated by blue light in the leaves of terrestrial angiosperms. Red light has been shown to affect the spatial reorganization of F-actin in water plants, where chloroplast movements are closely connected with cytoplasmic streaming. The aim of the present study was to determine whether blue light, which triggers characteristic responses of chloroplasts, i.e. avoidance and accumulation, also influences F-actin organization in the mesophyll cells of Arabidopsis thaliana. Actin filaments in fixed mesophyll tissue were labelled with Alexa Fluor 488-conjugated phalloidin. The configuration of actin filaments, expressed as a form factor (4 pi x area/perimeter(2)), was determined for all actin formations which were measured in fluorescence confocal images. RESULTS: In the present study, we compare form-factor distributions and the median form factors for strong and weak, blue- and red-irradiated tissues. Spatial organization of the F-actin network did not undergo any changes which could be attributed specifically to blue light. Actin patterns were similar in blue-irradiated wild-type plants and phot2 (phototropin 2) mutants which lack the avoidance response of chloroplasts. However, significant differences in the shape and distribution of F-actin formations were observed between mesophyll cells of phot2 mutants irradiated with strong and weak red light. These differences were absent in wild-type leaves. CONCLUSIONS: Actin does not appear to be the main target for the blue-light chloroplast-orientation signal. The modes of actin involvement in chloroplast translocations are different in water and terrestrial angiosperms. The results suggest that co-operation occurs between blue- and red-light photoreceptors in the control of the actin cytoskeleton architecture in Arabidopsis.     

Chloroplast movement: dissection of events downstream of photo- and mechano-perception

The study of chloroplast photorelocation movement is progressing rapidly now that mutants for chloroplast movement have become available in Arabidopsis thaliana. However, mechanistic approaches in cell biology still stand to elucidate the mechanisms and regulations of such movement. The fern Adiantum capillus-veneris and the moss Physcomitrella patens are particularly suitable materials for analyzing the kinetics of intracellular chloroplast movement. In these plants, chloroplast movement is induced by red light as well as blue light, mediated by phytochrome and blue light receptor, respectively. In this paper, we review the unique force-generating system for chloroplast motility in P. patens. In addition to light-induced chloroplast movement, we also summarize mechanically induced chloroplast movement in these plants and the motility systems involved. Finally, the different dependency of mechano- and photo-relocation movement on external Ca²⁺ is discussed. Electronic Publication     

Phot2‐regulated relocation of NPH3 mediates phototropic response to high‐intensity blue light in Arabidopsis thaliana

Two redundant blue‐light receptors, known as phototropins (phot1 and phot2), influence a variety of physiological responses, including phototropism, chloroplast positioning, and stomatal opening in Arabidopsis thaliana. Whereas phot1 functions in both low‐ and high‐intensity blue light (HBL), phot2 functions primarily in HBL. Here, we aimed to elucidate phot2‐specific functions by screening for HBL‐insensitive mutants among mutagenized Arabidopsis phot1 mutants. One of the resulting phot2 signaling associated (p2sa) double mutants, phot1 p2sa2, exhibited phototropic defects that could be restored by constitutively expressing NON‐PHOTOTROPIC HYPOCOTYL 3 (NPH3), indicating that P2SA2 was allelic to NPH3. It was observed that NPH3‐GFP signal mainly localized to and clustered on the plasma membrane in darkness. This NPH3 clustering on the plasma membrane was not affected by mutations in genes encoding proteins that interact with NPH3, including PHOT1, PHOT2 and ROOT PHOTOTROPISM 2 (RPT2). However, the HBL irradiation‐mediated release of NPH3 proteins into the cytoplasm was inhibited in phot1 mutants and enhanced in phot2 and rpt2‐2 mutants. Furthermore, HBL‐induced hypocotyl phototropism was enhanced in phot1 mutants and inhibited in the phot2 and rpt2‐2 mutants. Our findings indicate that phot1 regulates the dissociation of NPH3 from the plasma membrane, whereas phot2 mediates the stabilization and relocation of NPH3 to the plasma membrane to acclimate to HBL.     

Phototropins and neochrome1 mediate nuclear movement in the fern Adiantum capillus-veneris

In gametophytic cells (prothalli) of the fern Adiantum capillus-veneris, nuclei as well as chloroplasts change their position according to light conditions. Nuclei reside on anticlinal walls in darkness and move to periclinal or anticlinal walls under weak or strong light conditions, respectively. Here we reveal that red light-induced nuclear movement is mediated by neochrome1 (neo1), blue light-induced movement is redundantly mediated by neo1, phototropin2 (phot2) and possibly phot1, and dark positioning of both nuclei and chloroplasts is mediated by phot2. Thus, both the nuclear and chloroplast photorelocation movements share common photoreceptor systems.     

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.     

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.