Plant Blue-light Receptors Research Articles

AmiRNAi silencing of FaCRY2 delayed the growth of cultivated strawberry

As a plant blue light receptor, CRY2 plays pleotropic roles in regulating photomorphogenesis, flowering time and stress response. However, the function of CRY2 in the octoploid cultivated strawberry (Fragaria × ananassa Duch.) has not been identified. In this study, certain variation and functional divergences of the FaCRY2 protein were found in compared with its homologs in diploid model plants (AtCRY2 and SlCRY2). Quantitative RT-PCR analysis of the FaCRY2 showed the highest expression level in the leaf and flower tissues. The subcellular localization results indicate that the FaCRY2 was a nucleus-localized protein. The cry2 mutants, silenced by artificial micro-interfering RNA interference (AmiRNAi) using 'Benihoppe' strawberry as genetic background, showed a growth retardation, including dwarf phenotype, decreased free IAA content, delayed flowering time, deepened the lignification degree of petiole cells, decreased stomatal conductance and transpiration rate. RNA-seq analysis showed that there were 910 significantly differentially expressed genes (DEGs) between wild-type (WT) and cry2. They were mainly enriched in hormone regulation, cell development and stress response, etc. The decrease of IAA accumulation could be the main factor responsible for the delayed growth of cry2. Taken together, our results suggested the complex roles of FaCRY2 in the plant growth regulation in cultivated strawberry.

Phototropin-Inspired Chemoselective Synthesis of Unsymmetrical Disulfides: Aerobic Oxidative Heterocoupling of Thiols Using Flavin Photocatalysis.

Inspired by the photochemical mechanism of a plant blue-light receptor, a unique flavin-based photocatalytic system was developed for the chemoselective heterocoupling of two different thiols, which enabled the facile synthesis of unsymmetrical disulfides. Owing to the redox- and photo-organocatalysis of flavin, the coupling reaction took place under mild metal-free conditions and visible light irradiation with the use of air, which is recognized as the ideal green oxidant.

Domain Organization in Plant Blue-Light Receptor Phototropin2 of Arabidopsis thaliana Studied by Small-Angle X-ray Scattering.

Phototropin2 (phot2) is a blue-light (BL) receptor protein that regulates the BL-dependent activities of plants for efficient photosynthesis. Phot2 is composed of two light-oxygen-voltage sensing domains (LOV1 and LOV2) to absorb BL, and a kinase domain. Photo-activated LOV domains, especially LOV2, play a major role in photo-dependent increase in the phosphorylation activity of the kinase domain. The atomic details of the overall structure of phot2 and the intramolecular mechanism to convert BL energy to a phosphorylation signal remain unknown. We performed structural studies on the LOV fragments LOV1, LOV2, LOV2-linker, and LOV2-kinase, and full-length phot2, using small-angle X-ray scattering (SAXS). The aim of the study was to understand structural changes under BL irradiation and discuss the molecular mechanism that enhance the phosphorylation activity under BL. SAXS is a suitable technique for visualizing molecular structures of proteins in solution at low resolution and is advantageous for monitoring their structural changes in the presence of external physical and/or chemical stimuli. Structural parameters and molecular models of the recombinant specimens were obtained from SAXS profiles in the dark, under BL irradiation, and after dark reversion. LOV1, LOV2, and LOV2-linker fragments displayed minimal structural changes. However, BL-induced rearrangements of functional domains were noted for LOV2-kinase and full-length phot2. Based on the molecular model together with the absorption measurements and biochemical assays, we discuss the intramolecular interactions and domain motions necessary for BL-enhanced phosphorylation activity of phot2.

CIPK23 regulates blue light-dependent stomatal opening in Arabidopsis thaliana.

Phototropins (phot1 and phot2) are plant blue light receptor kinases that function to mediate phototropism, chloroplast movement, leaf flattening, and stomatal opening in Arabidopsis. Considerable progress has been made in understanding the mechanisms associated with phototropin receptor activation by light. However, the identities of phototropin signaling components are less well understood by comparison. In this study, we specifically searched for protein kinases that interact with phototropins by using an in vitro screening method (AlphaScreen) to profile interactions against an Arabidopsis protein kinase library. We found that CBL-interacting protein kinase 23 (CIPK23) interacts with both phot1 and phot2. Although these interactions were verified by in vitro pull-down and in vivo bimolecular fluorescence complementation assays, CIPK23 was not phosphorylated by phot1, as least in vitro. Mutants lacking CIPK23 were found to exhibit impaired stomatal opening in response to blue light but no deficits in other phototropin-mediated responses. We further found that blue light activation of inward-rectifying K+ (K+ in ) channels was impaired in the guard cells of cipk23 mutants, whereas activation of the plasma membrane H+ -ATPase was not. The blue light activation of K+ in channels was also impaired in the mutant of BLUS1, which is one of the phototropin substrates in guard cells. We therefore conclude that CIPK23 promotes stomatal opening through activation of K+ in channels most likely in concert with BLUS1, but through a mechanism other than activation of the H+ -ATPase. The role of CIPK23 as a newly identified component of phototropin signaling in stomatal guard cells is discussed.

Structural insights into BIC-mediated inactivation of Arabidopsis cryptochrome 2.

Cryptochromes (CRYs) are blue-light receptors in plants that harbor FAD as a cofactor and regulate various physiological responses. Photoactivated CRYs undergo oligomerization, which increases the binding affinity to downstream signaling partners. Despite decades of research on the activation of CRYs, little is known about how they are inactivated. Binding of blue-light inhibitors of cryptochromes (BICs) to CRY2 suppresses its photoactivation, but the underlying mechanism remains unknown. Here, we report crystal structures of CRY2N (CRY2 PHR domain) and the BIC2-CRY2N complex with resolutions of 2.7 and 2.5 Å, respectively. In the BIC2-CRY2N complex, BIC2 exhibits an extremely extended structure that sinuously winds around CRY2N. In this way, BIC2 not only restrains the transfer of electrons and protons from CRY2 to FAD during photoreduction but also interacts with the CRY2 oligomer to return it to the monomer form. Uncovering the mechanism of CRY2 inactivation lays a solid foundation for the investigation of cryptochrome protein function.

Phototropins Mediate Chloroplast Movement in Phalaenopsis aphrodite (Moth Orchid).

Chloroplast movement is important for plants to avoid photodamage and to perform efficient photosynthesis. Phototropins are blue light receptors in plants that function in chloroplast movement, phototropism, stomatal opening, and they also affect plant growth and development. In this study, full-length cDNAs of two PHOTOTROPIN genes, PaPHOT1 and PaPHOT2, were cloned from a moth orchid Phalaenopsis aphrodite, and their functions in chloroplast movement were investigated. Phylogenetic analysis showed that PaPHOT1 and PaPHOT2 orthologs were highly similar to PHOT1 and PHOT2 of the close relative Phalaenopsis equestris, respectively, and clustered with monocots PHOT1 and PHOT2 orthologs, respectively. Phalaenopsis aphrodite expressed a moderate level of PaPHOT1 under low blue light of 5 μmol�m-2�s-1 (BL5) and a high levels of PaPHOT1 at >BL100. However, PaPHOT2 was expressed at low levels at <BL50 but expressed at high levels at > BL100. Analysis of light-induced chloroplast movements using the SPAD method indicated that orchid accumulated chloroplasts at <BL10. The chloroplast avoidance response was detectable at >BL25 and significant chloroplast avoidance movement was observed at >BL100. Virus-induced gene silencing of PaPHOTs in orchids showed decreased gene expression of PaPHOTs and reduced both chloroplast accumulation and avoidance responses. Heterologous expression of PaPHOT1 in Arabidopsis phot1phot2 double mutant recovered chloroplast accumulation response at BL5, but neither PaPHOT1 nor PaPHOT2 was able to restore mutant chloroplast avoidance at BL100. Overall, this study showed that phototropins mediate chloroplast movement in Phalaenopsis orchid is blue light-dependent but their function is slightly different from Arabidopsis which might be due to gene evolution.

Essential role of the A'α/Aβ gap in the N-terminal upstream of LOV2 for the blue light signaling from LOV2 to kinase in Arabidopsis photototropin1, a plant blue light receptor.

Phototropin (phot) is a blue light (BL) receptor in plants and is involved in phototropism, chloroplast movement, stomata opening, etc. A phot molecule has two photo-receptive domains named LOV (Light-Oxygen-Voltage) 1 and 2 in its N-terminal region and a serine/threonine kinase (STK) in its C-terminal region. STK activity is regulated mainly by LOV2, which has a cyclic photoreaction, including the transient formation of a flavin mononucleotide (FMN)-cysteinyl adduct (S390). One of the key events for the propagation of the BL signal from LOV2 to STK is conformational changes in a Jα-helix residing downstream of the LOV2 C-terminus. In contrast, we focused on the role of the A’α-helix, which is located upstream of the LOV2 N-terminus and interacts with the Jα-helix. Using LOV2-STK polypeptides from Arabidopsis thaliana phot1, we found that truncation of the A’α-helix and amino acid substitutions at Glu474 and Lys475 in the gap between the A’α and the Aβ strand of LOV2 (A’α/Aβ gap) to Ala impaired the BL-induced activation of the STK, although they did not affect S390 formation. Trypsin digested the LOV2-STK at Lys603 and Lys475 in a light-dependent manner indicating BL-induced structural changes in both the Jα-helix and the gap. The digestion at Lys603 is faster than at Lys475. These BL-induced structural changes were observed with the Glu474Ala and the Lys475Ala substitutes, indicating that the BL signal reached the Jα-helix as well as the A’α/Aβ gap but could not activate STK. The amino acid residues, Glu474 and Lys475, in the gap are conserved among the phots of higher plants and may act as a joint to connect the structural changes in the Jα-helix with the activation of STK.

ATP binding and aspartate protonation enhance photoinduced electron transfer in plant cryptochrome.

Cryptochromes are flavoproteins encountered in most vegetal and animal species. They play a role of blue-light receptors in plants and in invertebrates. The putative resting state of the FAD cofactor in these proteins is its fully oxidized form, FADox. Upon blue-light excitation, the isoalloxazine ring (ISO) may undergo an ultrafast reduction by a nearby tryptophan residue W400. This primary reduction triggers a cascade of electron and proton transfers, ultimately leading to the formation of the FADH° radical. A recent experimental study has shown that the yield of FADH° formation in Arabidopsis cryptochrome can be strongly modulated by ATP binding and by pH, affecting the protonation state of D396 (proton donor to FAD°(-)). Here we provide a detailed molecular analysis of these effects by means of combined classical molecular dynamics simulations and time-dependent density functional theory calculations. When ATP is present and D396 protonated, FAD remains in close contact with W400, thereby enhancing electron transfer (ET) from W400 to ISO*. In contrast, deprotonation of D396 and absence of ATP introduce flexibility to the photoactive site prior to FAD excitation, with the consequence of increased ISO-W400 distance and diminished tunneling rate by almost two orders of magnitude. We show that under these conditions, ET from the adenine moiety of FAD becomes a competitive relaxation pathway. Overall, our data suggest that the observed effects of ATP and pH on the FAD photoreduction find their roots in the earliest stage of the photoreduction process; i.e., ATP binding and the protonation state of D396 determine the preferred pathway of ISO* relaxation.

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 Ca(2+) concentration ([Ca(2+)]cyt) in Arabidopsis (Arabidopsis thaliana) hypocotyls and that the increase in [Ca(2+)]cyt was primarily attributed to phot2. Pharmacological and genetic experiments illustrated that HBL-induced Ca(2+) increases were modulated differently by phot1 and phot2. Phot2 mediated the HBL-induced increase in [Ca(2+)]cyt mainly by an inner store-dependent Ca(2+)-release pathway, not by activating plasma membrane Ca(2+) channels. Further analysis showed that the increase in [Ca(2+)]cyt was possibly responsible for HBL-induced hypocotyl phototropism. An inhibitor of auxin efflux carrier exhibited significant inhibitions of both phototropism and increases in [Ca(2+)]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 Ca(2+) signaling-related HBL modulates hypocotyl phototropic responses.

SCF(FBXL3) ubiquitin ligase targets cryptochromes at their cofactor pocket.

The flavoprotein cryptochromes (CRYs) act as blue-light receptors in plants and insects, but perform light-independent functions at the core of the mammalian circadian clock. To drive clock oscillations, mammalian CRYs associate with the Period proteins (PERs) and together inhibit the transcription of their own genes. The SCFFbxl3 ubiquitin ligase complex controls this negative feedback loop by promoting CRY ubiquitylation and degradation. Yet, the molecular mechanisms of their interactions and the functional role of flavin adenine dinucleotide (FAD) binding in CRYs remain poorly understood. Here we report crystal structures of mammalian CRY2 in its apo, FAD-bound, and Fbxl3-Skp1-complexed forms. Distinct from other cryptochromes of known structures, mammalian CRY2 binds FAD dynamically with an open cofactor pocket. Strikingly, the F-box protein Fbxl3 captures CRY2 by simultaneously occupying its FAD-binding pocket with a conserved C-terminal tail and burying its PER-binding interface. This novel F-box protein-substrate bipartite interaction is susceptible to disruption by both FAD and PERs, suggesting a new avenue for pharmacological targeting of the complex and a multifaceted regulatory mechanism of CRY ubiquitylation.

1SAP-03 植物の青色光受容体phototropinの全長でのシグナリング機構(1SAP 新学術領域「植物の環境感覚 : 刺激受容から細胞応答まで」共催,カラフルな植物光環境感覚タンパク質,シンポジウム,日本生物物理学会第51回年会(2013年度))

1SAP-03 植物の青色光受容体phototropinの全長でのシグナリング機構(1SAP 新学術領域「植物の環境感覚 : 刺激受容から細胞応答まで」共催,カラフルな植物光環境感覚タンパク質,シンポジウム,日本生物物理学会第51回年会(2013年度))

Design and Engineering of a Light-Activated Potassium Channel

Optogenetics uses light-activated ion channels to manipulate electrical signals of cells with high spatio-temporal precision. The available palette of optogenetics tools so far includes activators and inhibitors. The sodium permeant channelrhodopsin-2 for example depolarizes the membrane and increases excitability. Light-activated chloride and proton pumps function as inhibitors of excitability because they hyperpolarize the cell. To widen the repertoire of optogenetic tools, the search is on for a light-activated potassium (K+) channel, which would enrich the efficacy of inhibitory switches. In the present work, we will describe our strategy to engineer, by a synthetic approach, a genetically encoded light-activated K+ channel. To this end, we have fused the LOV (light oxygen voltage) domain of the plant blue-light receptor phototropin, to the viral K+ channel Kcv. The functional properties of the resulting chimeric channels BLINK (blue light induced K+ channel) were optimized by a combination of rational and irrational design using a high-throughput yeast-based screening system. We have so far selected two promising prototype channels, which show opposite regulation by blue light:, BLINK1 is activated, while BLINK2 inhibited by blue light. Both prototype channels are functional when expressed in Xenopus oocytes and we will discuss their biophysical properties in the context of future optogenetics applications.

Photosensitivity of Kinase Activation by Blue Light Involves the Lifetime of a Cysteinyl-Flavin Adduct Intermediate, S390, in the Photoreaction Cycle of the LOV2 Domain in Phototropin, a Plant Blue Light Receptor

Phototropin (phot) is a light-regulated protein kinase that mediates a variety of photoresponses in plants, such as phototropism, chloroplast positioning, and stomata opening. Arabidopsis has two homologues, phot1 and phot2, that share physiological functions depending on light intensity. A phot molecule has two photoreceptive light oxygen voltage-sensing domains, LOV1 and LOV2, and a Ser/Thr kinase domain. The LOV domains undergo a photocycle upon blue light (BL) stimulation, including transient adduct formation between the chromophore and a conserved cysteine (S390 intermediate) that leads to activation of the kinase. To uncover the mechanism underlying the photoactivation of the kinase, we have introduced a kinase assay system composed of a phot1 LOV2-linker-kinase polypeptide as a light-regulated kinase and its N-terminal polypeptide as an artificial substrate (Okajima, K., Matsuoka, D., and Tokutomi, S. (2011) LOV2-linker-kinase phosphorylates LOV1-containing N-terminal polypeptide substrate via photoreaction of LOV2 in Arabidopsis phototropin1. FEBS Lett. 585, 3391-3395). In the present study, we extended the assay system to phot2 and compared the photochemistry and kinase activation by BL between phot1 and phot2 to gain insight into the molecular basis for the different photosensitivities of phot1 and phot2. Photosensitivity of kinase activation by BL and the lifetime of S390 of phot1 were 10 times higher and longer, respectively, than those of phot2. This correlation was confirmed by an amino acid substitution experiment with phot1 to shorten the lifetime of S390. The present results demonstrated that the photosensitivity of kinase activation in phot involves the lifetime of S390 in LOV2, suggesting that the lifetime is one of the key factors for the different photosensitivities observed for phot1 and phot2.

Blue and green are frequently seen: responses of seeds to short- and mid-wavelength light

AbstractSeeds have long been a model system for studying the intricacies of phytochrome-mediated light perception and signalling. However, very little is known about how they perceive blue and green light. Cryptochromes and phototropins, the major blue-light receptors in plants, are increasingly well-studied in vegetative tissues, but their role in light perception in seeds largely remains a mystery. Green light elicits a number of responses in plants that cannot be explained by the action of any of the known photoreceptors, and some seeds are apparently also capable of perceiving green light. Here, the responses of seeds to blue and green light are collated from a thorough examination of the literature and considered from the perspective of the potential photoreceptor(s) mediating them. Knowledge of how seeds perceive wavelengths that are suboptimal for phytochrome activation could help to improve germination and seedling establishment for both crop and native species.

LOV2-linker-kinase phosphorylates LOV1-containing N-terminal polypeptide substrate via photoreaction of LOV2 in Arabidopsis phototropin1

Phototropin is a blue light receptor in plants and is thought to be a light-regulated protein kinase. Previously, we defined the role of the photoreceptive domains, LOV1 and 2, in the light activation of the kinase in Arabidopsis phototropin2 (phot2) [1]. In this study, photoregulation of the kinase in phototropin1 (phot1) was studied using LOV2-linker-kinase polypeptide. We designed a new substrate consisting of the N-terminal part of the phot1 with autophosphorylation sites. The LOV2-linker-kinase had the same spectroscopic properties as those of the LOV2 core and phosphorylated the substrate in a light-dependent manner. Amino acid substitution experiments proved that the phosphorylation comes from the activation of the kinase via photoreaction of LOV2.Structured summary of protein interactions:AtPhot1-LOV2 phosphorylatesAtPhot1-Nt by protein kinase assay (View Interaction: 1, 2, 3)

FLOWERING LOCUS T Regulates Stomatal Opening

Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange for photosynthesis in response to light, CO(2), and phytohormone abscisic acid. Phototropins (phot1 and phot2) are plant blue-light receptor kinases and mediate stomatal opening via activation of the plasma membrane H(+)-ATPase. However, the signaling mechanism from phototropins to the H(+)-ATPase has yet to be determined. Here, we show that FLOWERING LOCUS T (FT) is expressed in guard cells and regulates stomatal opening. We isolated an scs (suppressor of closed-stomata phenotype in phot1 phot2) 1-1 mutant of Arabidopsis thaliana and showed that scs1-1 carries a novel null early flowering 3 (elf3) allele in a phot1 phot2 background. scs1-1 (elf3 phot1 phot2 triple mutant) had an open-stomata phenotype with high H(+)-ATPase activity and showed increased levels of FT mRNA in guard cells. Transgenic plants overexpressing FT in guard cells showed open stomata, whereas a loss-of-function FT allele, ft-1, exhibited closed stomata and failed to activate the H(+)-ATPase in response to blue light. Our results define a new cell-autonomous role for FT and demonstrate that the flowering time genes ELF3 and FT are involved in the regulation of H(+)-ATPase by blue light in guard cells.

3Q1510 Signaling mechanisms of phototropin, plant blue light receptor(Photobiology : Vision & Photoreception4,The 49th Annual Meeting of the Biophysical Society of Japan)

3Q1510 Signaling mechanisms of phototropin, plant blue light receptor(Photobiology : Vision & Photoreception4,The 49th Annual Meeting of the Biophysical Society of Japan)

青色光受容体フォトトロピンの"Flippase-kinase"としての役割の解明

青色光受容体フォトトロピンの"Flippase-kinase"としての役割の解明

A Wandering Pathway in Plant Biology: From Wildflowers to Phototropins to Bacterial Virulence

The author describes the somewhat convoluted pathway he followed from amateur taxonomy of Minnesota wildflowers to identification of the phototropin family of blue-light receptors. He also mentions individuals who were important in moving his career first into plant taxonomy, then plant development, and finally plant photobiology (and out of music). He emphasizes the many twists and turns a research career can take, including a few that lead to blind ends. He also emphasizes the oscillatory nature of his career-back and forth between the Atlantic and Pacific oceans (with occasional forays to Freiburg, Germany) and back and forth between red-light receptors and blue-light receptors. There is a short intermission in which he describes his longtime relationship with California's Henry W. Coe State Park. Finally, he relates how he followed the unlikely pathway from plant blue-light receptors to a blue-light receptor required to maximize virulence of a bacterial animal pathogen.

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.