Mapping of low- and high-fluence autophosphorylation sites in phototropin 1

Phototropins, originally detected by their blue light-dependent autophosphorylation, are plant photoreceptors involved in several blue light responses such as phototropism, chloroplast relocation, leaf expansion, rapid inhibition of hypocotyl growth, and stomatal opening. Three domains have been identified in phototropin sequences, two chromophore binding domains (LOV1 and LOV2) and a kinase domain. We describe here two additional domains, the N-terminus upstream of LOV1 and the hinge region between LOV1 and LOV2, as the regions for autophosphorylation; the phosphorylation sites were identified by site-directed mutagenesis as S27, S30, S274, S300, S317, S325, S332, and S349 of the PHOT1a sequence of Avena sativa. Investigation of the autophosphorylation in vivo revealed that serines close to the LOV1 domain are phosphorylated at lower fluence of blue light than the serines close to the LOV2 domain. Recovery of phosphorylation in vivo during a dark period after saturating irradiation is caused by dephosphorylation rather than by degradation of the phosphorylated form and new synthesis of nonphosphorylated phototropin. The results were obtained by a combination of autophosphorylation of phototropin with phosphorylation of recombinant domains by protein kinase A, which turned out to have the same site specificity as the phototropin kinase, followed by proteolysis and separation of phosphopeptides. With the knowledge of the phosphorylation sites, the physiological and biochemical consequences of autophosphorylation can now be approached by site-directed mutagenesis of phototropins.     

蓝光调控的植物器官与细胞器运动

蓝光可诱导植物的器官和细胞器运动,从而更好地适应环境。这些运动反应包括向光反应(phototropism)、叶绿体运动(chloroplast movements)、气孔开放(stomatal opening)、细胞核定位(nuclei positioning)以及叶片伸展和定位(leaf expansion and positioning)。调控这些运动反应的主要蓝光受体是向光素(phototropins)。本文综述向光素的分子特性与感光机制,以及由向光素介导的各种器官及细胞器运动和相关信号转导途径的最新研究进展。     

The C-terminal kinase fragment of Arabidopsis phototropin 2 triggers constitutive phototropin responses

Phototropins mediate various blue-light responses such as phototropism, chloroplast relocation, stomatal opening and leaf flattening in plants. Phototropins are hydrophilic chromoproteins that are mainly bound to the plasma membrane. One of two phototropins in Arabidopsis thaliana, phot2, associates with the Golgi apparatus in a light-dependent manner. In this study, we analyzed the biological activities of the N-terminal photosensory and C-terminal kinase domains of phot2. For this purpose, these domains were fused to green fluorescent protein (GFP) and ectopically expressed in the wild-type and a phot1 phot2 double mutant of Arabidopsis. The kinase domain fused to GFP (P2CG) was localized to the plasma membrane and the Golgi apparatus, whereas the photosensory domain fused to GFP (P2NG) was uniformly localized in the cytosol. Hence, the kinase domain rather than the photosensory domain is responsible for the membrane association. Interestingly, the P2CG plants exhibited constitutive blue-light responses even in dark conditions, i.e. stomata were open and chloroplasts were in the avoidance position. By contrast, P2CG with a mutation that abolishes the kinase activity (P2C[D720/N]G) failed to exhibit these responses. phot2 kinase is therefore suggested to be correctly localized to functional sites in the cell and to trigger light signal transduction through its kinase activity. In contrast to P2CG, P2NG did not affect the phot2 responses, except for partial inhibition of the phototropic response caused by the endogenous phototropins.     

Phototropin-mediated perception of light direction in leaves regulates blade flattening

One conserved feature among angiosperms is the development of flat thin leaves. This developmental pattern optimizes light capture and gas exchange. The blue light (BL) receptors phototropins are required for leaf flattening, with the null phot1phot2 mutant showing curled leaves in Arabidopsis (Arabidopsis thaliana). However, key aspects of their function in leaf development remain unknown. Here, we performed a detailed spatiotemporal characterization of phototropin function in Arabidopsis leaves. We found that phototropins perceive light direction in the blade, and, similar to their role in hypocotyls, they control the spatial pattern of auxin signaling, possibly modulating auxin transport, to ultimately regulate cell expansion. Phototropin signaling components in the leaf partially differ from hypocotyls. Moreover, the light response on the upper and lower sides of the leaf blade suggests a partially distinct requirement of phototropin signaling components on each side. In particular, NON PHOTOTROPIC HYPOCOTYL 3 showed an adaxial-specific function. In addition, we show a prominent role of PHYTOCHROME KINASE SUBSTRATE 3 in leaf flattening. Among auxin transporters, PIN-FORMED 3,4,7 and AUXIN RESISTANT 1 (AUX1)/LIKE AUXIN RESISTANT 1 (LAX1) are required for the response while ABCB19 has a regulatory role. Overall, our results show that directional BL perception by phototropins is a key aspect of leaf development, integrating endogenous and exogenous signals.

Phototropins perceive light direction in the leaf and control the auxin signaling pattern to regulate blade flattening.     

Domain Swapping to Assess the Mechanistic Basis of Arabidopsis Phototropin 1 Receptor Kinase Activation and Endocytosis by Blue Light

Phototropins (phot1 and phot2) are plasma membrane–associated receptor kinases that respond specifically to blue and UV wavelengths. In addition to a C-terminal Ser/Thr kinase domain, phototropins contain two N-terminal chromophore binding LOV domains that function as photoswitches to regulate a wide range of enzymatic activities in prokaryotes and eukaryotes. Through domain swapping, we show that the photochemical properties of Arabidopsis thaliana phot1 rely on interactions between LOV1 and LOV2, which are facilitated by their intervening linker sequence. Functional analysis of domain-swap proteins supports a mechanism whereby LOV2 acts as a dark-state repressor of phot1 activity both in vitro and in vivo. Moreover, we find a photoactive role for LOV1 in arresting chloroplast accumulation at high light intensities. Unlike LOV2, LOV1 cannot operate as a dark-state repressor, resulting in constitutive receptor autophosphorylation and accelerated internalization from the plasma membrane. Coexpression of active and inactive forms of phot1 demonstrates that autophosphorylation can occur intermolecularly, independent of LOV1, via light-dependent receptor dimerization in vivo. Indeed, transphosphorylation is sufficient to promote phot1 internalization through a clathrin-dependent endocytic pathway triggered primarily by phosphorylation of Ser-851 within the kinase activation loop. The mechanistic implications of these findings in regard to light-driven receptor activation and trafficking are discussed.     

A transgene encoding a blue-light receptor, phot1, restores blue-light responses in the Arabidopsis phot1 phot2 double mutant

Phototropins (phot1 and phot2) are suggested to be multifunctional blue-light (BL) receptors mediating phototropism, chloroplast movement, stomatal opening, and leaf expansion. The Arabidpsis phot1 phot2 double mutant lacks all of these responses. To confirm the requirement of phototropins in BL responses, the Arabidopsis phot1 phot2 double mutant was transformed with PHOT1 cDNA and the phenotypic restoration was analysed in the transformants. It was found that all BL responses were restored, although differentially, by the transformation of the Arabidopsis phot1 phot2 double mutant with PHOT1 cDNA. The results showed that phot1 was an essential component for all these BL responses in planta, and that the cellular level of phot1 might determine the individual BL 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.     

Phototropin receptor kinase activation by blue light

Phototropins (phot1 and phot2) are blue light-activated serine/threonine protein kinases that function to mediate a variety of adaptive processes that serve to optimize the photosynthetic efficiency of plants and thereby promote their growth. Light sensing by the phototropins is mediated by a repeated motif located within the N-terminal region of the protein designated the LOV domain. Although phototropins possess two LOV photosensors (LOV1 and LOV2), recent biophysical and structure-function analyses clearly indicate that the LOV2 domain plays a predominant role in regulating phototropin kinase activity owing to specific protein changes that occur in response to LOV2 photoexcitation. In particular, the central β-sheet scaffold plays a role in propagating the photochemical signal generated from within LOV2 to protein changes at the surface that are necessary for kinase activation.Key words: phototropin, LOV domain, FMN, cysteinyl adduct, amphipathic helix, receptor autophosphoryation     

High pigment1 mutation negatively regulates phototropic signal transduction in tomato seedlings

Phototropins and phytochromes are the major photosensory receptors in plants and they regulate distinct photomorphogenic responses. The molecular mechanisms underlying functional interactions of phototropins and phytochromes remain largely unclear. We show that the tomato (Lycopersicon esculentum) phytochrome A deficient mutant fri lacks phototropic curvature to low fluence blue light, indicating requirement for phytochrome A for expression of phototropic response. The hp1 mutant that exhibits hypersensitive responses to blue light and red light reverses the impairment of second-positive phototropic response in tomato in phytochrome A-deficient background. Physiological analyses indicate that HP1 functions as a negative regulator of phototropic signal transduction pathway, which is removed via action of phytochrome A. The loss of HP1 gene product in frihp1 double mutant allows the unhindered operation of phototropic signal transduction chain, obviating the need for the phytochrome action. Our results also indicate that the role of phytochrome in regulating phototropism is restricted to low fluence blue light only, and at high fluence blue light, the phytochrome A-deficient fri mutant shows the normal phototropic response.     

Blue-light- and phosphorylation-dependent binding of a 14-3-3 protein to phototropins in stomatal guard cells of broad bean

Phototropins are blue-light (BL) receptor serine (Ser)/threonine kinases, and contain two light, oxygen, and voltage (LOV) domains, and are members of the PAS domain superfamily. They mediate phototropism, chloroplast movement, leaf expansion, and stomatal opening of higher plants in response to BL. In stomatal guard cells, genetic analysis has revealed that phototropins mediate activation of the plasma membrane H+-ATPase by phosphorylation and drive stomatal opening. However, biochemical evidence for the involvement of phototropins in the BL response of stomata is lacking. Using guard cell protoplasts, we showed that broad bean (Vicia faba) phototropins (Vfphots) were phosphorylated by BL, and that this phosphorylation of Vfphots reached to the maximum level earlier than that of the H+-ATPase. Phosphorylation of both Vfphots and H+-ATPase showed similar sensitivity to BL and were similarly suppressed by protein kinase and flavoprotein inhibitors. We found that a 14-3-3 protein was bound to Vfphots upon phosphorylation, and this binding occurred earlier than the H+-ATPase phosphorylation. Vfphots (Vfphot1a and Vfphot1b) were expressed in Escherichia coli, and phosphorylation sites were determined to be Ser-358 for Vfphot1a and Ser-344 for Vfphot1b, which are localized between LOV1 and LOV2. We conclude that Vfphots act as BL receptors in guard cells and that phosphorylation of a Ser residue between LOV1 and LOV2 and subsequent 14-3-3 protein binding are likely to be key steps of BL response in stomata. The binding of a 14-3-3 protein to Vfphot was found in etiolated seedlings and leaves in response to BL, suggesting that this event was common to phototropin-mediated responses.     

Mutational analysis of phototropin 1 provides insights into the mechanism underlying LOV2 signal transmission

Phototropins (phot1 and phot2) are blue light-activated serine/threonine protein kinases that elicit a variety of photoresponses in plants. Light sensing by the phototropins is mediated by two flavin mononucleotide (FMN)-binding domains, designated LOV1 and LOV2, located in the N-terminal region of the protein. Exposure to light results in the formation of a covalent adduct between the FMN chromophore and a conserved cysteine residue within the LOV domain. LOV2 photoexcitation is essential for phot1 function in Arabidopsis and is necessary to activate phot1 kinase activity through light-induced structural changes within a conserved alpha-helix situated C-terminal to LOV2. Here we have used site-directed mutagenesis to identify further amino acid residues that are important for phot1 activation by light. Mutagenesis of bacterially expressed LOV2 and full-length phot1 expressed in insect cells indicates that perturbation of the conserved salt bridge on the surface of LOV2 does not play a role in receptor activation. However, mutation of a conserved glutamine residue (Gln(575)) within LOV2, reported previously to be required to propagate structural changes at the LOV2 surface, attenuates light-induced autophosphorylation of phot1 expressed in insect cells without compromising FMN binding. These findings, in combination with double mutant analyses, indicate that Gln(575) plays an important role in coupling light-driven cysteinyl adduct formation from within LOV2 to structural changes at the LOV2 surface that lead to activation of the C-terminal kinase domain.     

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.     

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。     

Higher plants use LOV to perceive blue light

Higher plants use several classes of blue light receptors to modulate a wide variety of physiological responses. Among them, both the phototropins and members of the Zeitlupe (ZTL) family use light oxygen voltage (LOV) photosensory domains. In Arabidopsis, these families comprise phot1, phot2 and ZTL, LOV Kelch Protein 2 (LKP2), and Flavin-binding Kelch F-box1 (FKF1). It has now been convincingly shown that blue-light-induced autophosphorylation of the phot1 kinase domain is an essential step in signal transduction. Recent experiments also shed light on the partially distinct photosensory specificities of phot1 and phot2. Phototropin signaling branches rapidly following photoreceptor activation to mediate distinct responses such as chloroplast movements or phototropism. Light activation of the LOV domain in ZTL family members modulates their capacity to interact with GIGANTEA (GI) and their ubiquitin E3 ligase activity. A complex between GI and FKF1 is required to trigger the degradation of a repressor of CO (CONSTANS) expression and thus modulates flowering time. In contrast, light-regulated complex formation between ZTL and GI appears to limit the capacity of ZTL to degrade its targets, which are part of the circadian oscillator.     

Blue light-induced orientation movements of chloroplasts in higher plants: Recent progress in the study of their mechanisms

The discovery of phototropins, photoreceptors for chloroplast responses in Arabidopsis thaliana, brought about renewed interest in these blue light-controlled movements. Recent progress in research on their mechanisms in higher plants is briefly summarized. Phototropins mediate phototropism, chloroplast relocations and stomatal movements. Their functions are partially overlapping, with phot1 active predominantly in weak light and phot2 active in strong light. The accumulation response of chloroplasts appears to be mediated by phot1 and phot2 whereas the avoidance response is controlled by phot2. The role of Ca²⁺ as a potential intracellular messenger has been discussed in view of the recently demonstrated blue light-induced transient increases in the cytosolic Ca²⁺ mediated differently by phot1 and phot2. Differential inhibition of accumulation and avoidance responses by wortmannin, the inhibitor of phosphoinositide-3 kinases, in Lemna trisulca points to an important role of these enzymes in the signal transduction. A new, multi-domain component controlling chloroplast positioning and movement, CHUP1, encodes an actin-binding protein in Arabidopsis.     

Photochemical properties of the flavin mononucleotide-binding domains of the phototropins from Arabidopsis,rice, and Chlamydomonas reinhardtii

Phototropins (phot1 and phot2, formerly designated nph1 and npl1) are blue-light receptors that mediate phototropism, blue light-induced chloroplast relocation, and blue light-induced stomatal opening in Arabidopsis. Phototropins contain two light, oxygen, or voltage (LOV) domains at their N termini (LOV1 and LOV2), each a binding site for the chromophore flavin mononucleotide (FMN). Their C termini contain a serine/threonine protein kinase domain. Here, we examine the kinetic properties of the LOV domains of Arabidopsis phot1 and phot2, rice (Oryza sativa) phot1 and phot2, and Chlamydomonas reinhardtii phot. When expressed in Escherichia coli, purified LOV domains from all phototropins examined bind FMN tightly and undergo a self-contained photocycle, characterized by fluorescence and absorption changes induced by blue light (T. Sakai, T. Kagawa, M. Kasahara, T.E. Swartz, J.M. Christie, W.R. Briggs, M. Wada, K. Okada [2001] Proc Natl Acad Sci USA 98: 6969-6974; M. Salomon, J.M. Christie, E. Knieb, U. Lempert, W.R. Briggs [2000] Biochemistry 39: 9401-9410). The photocycle involves the light-induced formation of a cysteinyl adduct to the C(4a) carbon of the FMN chromophore, which subsequently breaks down in darkness. In each case, the relative quantum efficiencies for the photoreaction and the rate constants for dark recovery of LOV1, LOV2, and peptides containing both LOV domains are presented. Moreover, the data obtained from full-length Arabidopsis phot1 and phot2 expressed in insect cells closely resemble those obtained for the tandem LOV-domain fusion proteins expressed in E. coli. For both Arabidopsis and rice phototropins, the LOV domains of phot1 differ from those of phot2 in their reaction kinetic properties and relative quantum efficiencies. Thus, in addition to differing in amino acid sequence, the phototropins can be distinguished on the basis of the photochemical cycles of their LOV domains. The LOV domains of C. reinhardtii phot also undergo light-activated spectral changes consistent with cysteinyl adduct formation. Thus, the phototropin family extends over a wide evolutionary range from unicellular algae to higher plants.     

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.     

Phototropins and Their LOV Domains：Versatile Plant Blue-Light Receptors

The phototropins phot1 and phot2 are plant blue-light receptors that mediate phototropism, chloroplast movements, stomatal opening, leaf expansion, the rapid Inhibition of hypocotyl growth in etiolated seedlings, and possibly solar tracking by leaves in those species in which It occurs. The phototroplns are plasma membrane-associated hydrophilic proteins with two chromophore domains （designated LOV1 and LOV2 for their resemblance to domains In other signaling proteins that detect light, oxygen, or voltage） in their Nterminal half and a classic serine/threonlne kinase domain in their C-terminal half. Both chromophore domains bind flavin mononucleotide （FMN） and both undergo light-activated formation of a covalent bond between a nearby cystelne and the C（4a） carbon of the FMN to form the signaling state. LOV2-cystelnyl adduct formation leads to the release downstream of a tightly bound amphlpathlc α-helix, a step required for activation of the klnase function. This cysteinyl adduct then slowly decays over a matter of seconds or minutes to return the photoreceptor chromophore modules to their ground state. Functional LOV2 is required for light-activated phosphorylation and for various blue-light responses mediated by the phototroplns. The function of LOV1 is still unknown, although It may serve to modulate the signal generated by LOV2. The LOV domain Is an ancient chromophore module found In a wide range of otherwise unrelated proteins In fungi and prokaryotes, the latter Including cyanobacterla, eubacterla, and archaea. Further general reviews on the phototropins are those by Celaya and Liscum （2005） and Christie and Briggs （2005）.