Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function

Phototropins (phot1 and phot2) are autophosphorylating serine/threonine kinases that function as photoreceptors for phototropism, light-induced chloroplast movement, and stomatal opening in Arabidopsis. The N-terminal region of phot1 and phot2 contains two specialized PAS domains, designated LOV1 and LOV2, which function as binding sites for the chromophore flavin mononucleotide (FMN). Both LOV1 and LOV2 undergo a self-contained photocycle, which involves the formation of a covalent adduct between the FMN chromophore and a conserved active-site cysteine residue (Cys39). Replacement of Cys39 with alanine abolishes the light-induced photochemical reaction of LOV1 and LOV2. Here we have used the Cys39Ala mutation to investigate the role of LOV1 and LOV2 in regulating phototropin function. Photochemical analysis of a bacterially expressed LOV1 + LOV2 fusion protein indicates that LOV2 functions as the predominant light-sensing domain for phot1. LOV2 also plays a major role in mediating light-dependent autophosphorylation of full-length phot1 expressed in insect cells and transgenic Arabidopsis. Moreover, photochemically active LOV2 alone in full-length phot1 is sufficient to elicit hypocotyl phototropism in transgenic Arabidopsis, whereas photochemically active LOV1 alone is not. Further photochemical and biochemical analyses also indicate that the LOV1 and LOV2 domains of phot2 exhibit distinct roles. The significance for the different roles of the phototropin LOV domains is discussed.     

Second positive phototropism results from coordinated co-action of the phototropins and cryptochromes

Phototropism and hypocotyl growth inhibition are modulated by the coaction of different blue-light photoreceptors and their signaling pathways. How seedlings integrate the activities of the different blue-light photoreceptors to coordinate these hypocotyl growth responses is still unclear. We have used time-lapse imaging and a nontraditional mathematical approach to conduct a detailed examination of phototropism in wild-type Arabidopsis and various blue-light photoreceptor mutants. Our results indicate that high fluence rates of blue light (100 micro mol m(-)(2) s(-)(1)) attenuate phototropism through the coaction of the phototropin and cryptochrome blue-light photoreceptors. In contrast, we also demonstrate that phototropins and cryptochromes function together to enhance phototropism under low fluence rates (<1.0 micro mol m(-)(2) s(-)(1)) of blue light. Based on our results, we hypothesize that phototropins and cryptochromes regulate phototropism by coordinating the balance between stimulation and inhibition of growth of the hypocotyl depending on the fluence rate of 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.     

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.     

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.     

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...     

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.     

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.     

Plant blue-light receptors

Plants have several blue-light receptors, which regulate different aspects of growth and development. Recent studies have identified three such receptors: cryptochrome 1, cryptochrome 2 and phototropin. Cryptochromes 1 and 2 are photolyase-like receptors that regulate hypocotyl growth and flowering time; phototropin mediates phototropism in response to blue light. In addition, phytochrome A has also been found to mediate various blue-light responses. Although the signal-transduction mechanisms of blue-light receptors remain largely unclear, phototropin is probably a protein kinase that regulates cytoplasmic calcium concentrations, whereas the cryptochromes might regulate anion-channel activity and changes in gene expression.     

Phototropins 1 and 2: versatile plant blue-light receptors

Blue and ultraviolet-A light regulate a wide range of responses in plants, including phototropism, chloroplast migration and stomatal opening. However, the photoreceptors for these light responses have been identified only recently. The phototropins (phot1 and phot2) represent a new class of receptor kinases that appear to be exclusive to plants. Recent genetic analysis has shown that phot1 and phot2 exhibit partially overlapping functions in mediating phototropism, chloroplast migration, and stomatal opening in Arabidopsis. Although significant progress has been made in understanding the early photochemical and biochemical events that follow phototropin excitation, the details of how this excitation activates such different responses remain to be elucidated.     

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.     

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.     

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     

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.     

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.     

Genetic separation of phototropism and blue light inhibition of stem elongation

Blue light-induced regulation of cell elongation is a component of the signal response pathway for both phototropic curvature and inhibition of stem elongation in higher plants. To determine if blue light regulates cell elongation in these responses through shared or discrete pathways, phototropism and hypocotyl elongation were investigated in several blue light response mutants in Arabidopsis thaliana. Specifically, the blu mutants that lack blue light-dependent inhibition of hypocotyl elongation were found to exhibit a normal phototropic response. In contrast, a phototropic null mutant (JK218) and a mutant that has a 20- to 30-fold shift in the fluence dependence for first positive phototropism (JK224) showed normal inhibition of hypocotyl elongation in blue light. F1 progeny of crosses between the blu mutants and JK218 showed normal phototropism and inhibition of hypocotyl elongation, and approximately 1 in 16 F2 progeny were double mutants lacking both responses. Thus, blue light-dependent inhibition of hypocotyl elongation and phototropism operate through at least some genetically distinct components.     

Unexpected roles for cryptochrome 2 and phototropin revealed by high-resolution analysis of blue light-mediated hypocotyl growth inhibition

Blue light (BL) rapidly and strongly inhibits hypocotyl elongation during the photomorphogenic response known as de-etiolation, the transformation of a dark-grown seedling into a pigmented, photoautotrophic organism. In Arabidopsis thaliana, high-resolution studies of hypocotyl growth accomplished by computer-assisted electronic image capture and analysis revealed that inhibition occurs in two genetically independent phases, the first beginning within 30 sec of illumination. The present work demonstrates that phototropin (nph1), the photoreceptor responsible for phototropism, is largely responsible for the initial, rapid inhibition. Signaling from phototropin during the curvature response is dependent upon interaction with NPH3, but the results presented here demonstrate that NPH3 is not necessary for phototropin-dependent growth inhibition. Activation of anion channels, which transiently depolarizes the plasma membrane within seconds of BL, is an early event in the cryptochrome signaling pathway leading to a phase of growth inhibition that replaces the transient phototropin-dependent phase after approximately 30 min of BL. Surprisingly, cry1 and cry2 were found to contribute equally and non-redundantly to anion-channel activation and to growth inhibition between 30 and 120 min of BL. Inspection of the inhibition kinetics displayed by nph1 and nph1cry1 mutants revealed that the cryptochrome phase of inhibition is delayed in seedlings lacking phototropin. This result indicates that BL-activation of phototropin influences cryptochrome signaling leading to growth inhibition. Mutations in the NPQ1 gene, which inhibit BL-induced stomatal opening, do not affect any aspect of the growth inhibition within the first 120 min examined here, and NPQ1 does not affect the activation of anion channels.     

向光素调节植物向光性及其与光敏色素/隐花色素的相互关系

蓝光受体向光素(PHOT1/PHOT2)调节蓝光诱导的植物运动反应, 包括植物向光性、叶绿体运动、气孔运动和叶片伸展等。其中, 向光素介导的植物向光性能够促使植物弯向光源, 确保其以最佳取向捕获光源, 优化光合作用。光敏色素和隐花色素作为光受体也参与植物的向光性调节。该文综述了向光素介导的拟南芥(Arabidopsis thaliana)下胚轴向光弯曲信号转导及其与光敏色素、隐花色素协同作用的分子机制, 以期为改造植物光捕获能力及提高光利用效率提供理论基础。