Biomedical subjects

Pill-Soon Song

Publications and source records attributed to Pill-Soon Song.

18 recordsLinked to original sources

PubMed2006

Differential interactions of phytochrome A (Pr vs. Pfr) with monoclonal antibodies probed by a surface plasmon resonance technique.

Phytochromes are red- and far-red light-reversible photoreceptors for photomorphogenesis in plants. Phytochrome A is a dimeric chromopeptide that mediates very low fluence and high irradiance responses. To analyze the surface properties of phytochrome A (phyA), the epitopes of 21 anti-phyA monoclonal antibodies were determined by variously engineered recombinant phyA proteins and the dissociation constants of seven anti-phyA monoclonal antibodies with phyA were measured using a surface plasmon resonance (SPR)-based resonant mirror biosensor (IAsys). Purified oat phyA was immobilized on the sensor surface using a carboxymethyl dextran cuvette in advance, and the interactions of each chosen monoclonal antibody against phyA in either red light absorbing form (Pr) or far-red light absorbing form (Pfr) at different concentrations were monitored. The binding profiles were analyzed using the FAST Fit program of IAsys. The resultant values of dissociation constants clearly demonstrated the differential affinities between the phyA epitopes and the monoclonal antibodies dependent upon Pr vs. Pfr conformations. Monoclonal antibody mAP20 preferentially recognized the epitope at amino acids 653-731 in the Pr form, whereas mAA02, mAP21 and mAR07/mAR08 displayed preferential affinities for the Pfr's surfaces at epitopes 494-601 (the hinge region between the N- and C-terminal domains), 601-653 (hinge in PASI domain), and 772-1128 (C-terminal domain), respectively. The N-terminal extension (1-74) was not recognized by mAP09 and mAP15, suggesting that the N-terminal extreme is not exposed in the native conformation of phyA. On the other hand, the C-terminal domain becomes apparently exposed on Pr-to-Pfr phototransformation, suggesting an inter-domain cross-talk. The use of surface plasmon resonance spectroscopy offers a new approach to study the surface properties of phytochromes associated with the photoreversible structural changes, as well as for the study of protein-protein interactions of phytochromes with their interacting proteins involved in light signaling events in plants.

Antibodies, Monoclonal↗

PubMed2006

Time-resolved detection of conformational changes in oat phytochrome A: time-dependent diffusion.

Conformational changes in oat phytochrome A (phy) in solution after photoexcitation of the red-absorbing form (Pr) were studied in time-domain by the pulsed laser-induced transient grating technique. It was found that the diffusion coefficient (D) of far-red-absorbing form (Pfr) of large phy (1.3 x 10(-11) m(2) s(-1)) is markedly reduced compared with that of Pr (5.8 x 10(-11) m(2) s(-1)). This large reduction indicates that the conformation of Pfr is significantly changed from that of Pr, so that the intermolecular interaction with water molecules increases. This change completes within 1 ms after the photoexcitation. On the other hand, D of Pr of intact phy (4.1 x 10(-11) m(2) s(-1)) first decreases upon photoexcitation to 0.89 x 10(-11) m(2) s(-1) within 1 ms and then gradually increases with a time constant of 100 ms to the value of Pfr, 1.7 x 10(-11) m(2) s(-1). This slower phase suggests that the conformation of the N-terminal region changes with 100 ms to decrease the intermolecular interaction with water after a global change in the large phy region. The increase of D was interpreted in terms of alpha-helix formation in the Pfr form from the random coil structure in the Pr form.

Avena↗

PubMed2006

Autophosphorylation of Arabidopsis nucleoside diphosphate kinase 2 occurs only on its active histidine residue.

Arabidopsis nucleoside diphosphate kinase 2 (NDPK2) is a component in the phytochrome-mediated light signaling. In the present study, its autophosphorylation was investigated. Acid-stable and alkali-stable phosphorylated residues were analyzed under two different conditions. Results revealed that NDPK2 is phosphorylated only on its active histidine residue His197 and the presence of serine/threonine phosphorylation is an experimental artifact due to the harsh condition applied in the treatment of the phosphorylated protein sample. To resolve the controversy of whether serine/threonine phosphorylation of NDPK occurs as has been suggested by other NDPK studies, NDPK2 putative phosphorylation site mutants were generated and examined. No serine/threonine phosphorylation was identified in NDPK2 or implicated in its enzymatic activity. Further studies indicated that the low enzymatic activity and autophosphorylation level of NDPK2 mutant S199A are shown to be due to a damaged H-bonding with the active histidine residue His197 in the nucleotide-binding pocket. In addition, NDPK2 Kpn loop mutant T182A was found to possess an extremely low enzymatic activity and almost no autophosphorylation, suggesting the importance of the oligomeric states of NDPK2 in NDPK2 functioning.

Arabidopsis↗

PubMed2005

A possible role for NDPK2 in the regulation of auxin-mediated responses for plant growth and development.

Auxin plays many crucial roles in the course of plant growth and development, such as hook opening, leaf expansion and inhibition of mesocotyl elongation. Although its mechanism of action has not been clarified at the molecular level, recent studies have indicated that auxin triggers the induction of a number of genes known as primary auxin-responsive genes. Hence, the identification of the regulatory components in auxin-mediated cellular responses would help to elucidate the mechanism of the action of this hormone in plant growth and development. NDPK2 encodes a nucleoside diphosphate kinase 2 (NDPK2) in Arabidopsis. We aim to elucidate the possible role of NDPK2 in auxin-related cellular processes, in view of the finding that a ndpk2 mutant displays developmental defects associated with auxin. Interestingly, the ndpk2 mutant exhibits defects in cotyledon development and increased sensitivity to an inhibitor of polar auxin transport (naphthylphthalamic acid; NPA). Consistent with this phenotype, the transcript levels of specific auxin-responsive genes were reduced in the ndpk2 mutant plants treated with auxin. The amount of auxin transported from the shoot apex to the shoot/root transition zone of ndpk2 mutant plants was increased, compared with that in the wild-type plants. These results collectively suggest that NDPK2 appears to participate in auxin-regulated processes, partly through the modulation of auxin transport.

Gene Expression↗

PubMed2005

Phytochrome phosphorylation in plant light signaling.

Reversible protein phosphorylation is a switching mechanism used in eukaryotes to regulate various cellular signalings. In plant light signaling, sophisticated photosensory receptor systems operate to modulate growth and development. The photoreceptors include phytochromes, cryptochromes and phototropins. Despite considerable progresses in defining the photosensory roles of these photoreceptors, the primary biochemical mechanisms by which the photoreceptor molecules transduce the perceived light signals into cellular responses remain to be elucidated. The signal-transducing photoreceptors in plants are all phosphoproteins and/or protein kinases, suggesting that light-dependent protein phosphorylation and dephosphorylation play important roles in the function of the photoreceptors. This review focuses on the role of phytochromes' reversible phosphorylation involved in the light signal transduction in plants.

Light↗

PubMed2005

Phytochrome-specific type 5 phosphatase controls light signal flux by enhancing phytochrome stability and affinity for a signal transducer.

Environmental light information such as quality, intensity, and duration in red (approximately 660 nm) and far-red (approximately 730 nm) wavelengths is perceived by phytochrome photoreceptors in plants, critically influencing almost all developmental strategies from germination to flowering. Phytochromes interconvert between red light-absorbing Pr and biologically functional far-red light-absorbing Pfr forms. To ensure optimal photoresponses in plants, the flux of light signal from Pfr-phytochromes should be tightly controlled. Phytochromes are phosphorylated at specific serine residues. We found that a type 5 protein phosphatase (PAPP5) specifically dephosphorylates biologically active Pfr-phytochromes and enhances phytochrome-mediated photoresponses. Depending on the specific serine residues dephosphorylated by PAPP5, phytochrome stability and affinity for a downstream signal transducer, NDPK2, were enhanced. Thus, phytochrome photoreceptors have developed an elaborate biochemical tuning mechanism for modulating the flux of light signal, employing variable phosphorylation states controlled by phosphorylation and PAPP5-mediated dephosphorylation as a mean to control phytochrome stability and affinity for downstream transducers.

Arabidopsis↗

PubMed2004

NDPK2 as a signal transducer in the phytochrome-mediated light signaling.

Nucleoside-diphosphate kinase (NDPK) 2 in Arabidopsis has been identified as a phytochrome-interacting protein by using the C-terminal domain of phytochrome A (PhyA) as the bait in yeast two-hybrid screening. The far-red light-absorbing form of phytochrome (Pfr) A stimulates NDPK2 gamma-phosphate exchange activity in vitro. To better understand the multiple functions of NDPK and its role in phytochrome-mediated signaling, we characterized the interaction between phytochrome and NDPK2. Domain studies revealed that PER-ARNT-SIM domain A in the C-terminal domain of phytochrome is the binding site for NDPK2. Additionally, phytochrome recognizes both the NDPK2 C-terminal fragment and the NDPK2 hexameric structure to fulfill its binding. To illustrate the mechanism of how the Pfr form of phytochrome stimulates NDPK2, His-197-surrounding residue mutants were made and tested. Results suggested that the H-bonding with His-197 inside the nucleotide-binding pocket is critical for NDPK2 functioning. The pH dependence profiles of NDPK2 indicated that mutants with different activities from the wild type have different pK(a) values of His-197 and that NDPK2 hyperactive mutants possess lower pK(a) values. Because a lower pK(a) value of His-197 accelerates NDPK2 autophosphorylation and the phospho-transfer between the phosphorylated NDPK2 and its kinase substrate, we concluded that the Pfr form of phytochrome stimulates NDPK2 by lowering the pK(a) value of His-197.

Adenosine Triphosphate↗

PubMed2004

Structural analysis of Arabidopsis thaliana nucleoside diphosphate kinase-2 for phytochrome-mediated light signaling.

In plants, nucleoside diphosphate kinases (NDPKs) play a key role in the signaling of both stress and light. However, little is known about the structural elements involved in their function. Of the three NDPKs (NDPK1-NDPK3) expressed in Arabidopsis thaliana, NDPK2 is involved in phytochrome-mediated signal transduction. In this study, we found that the binding of dNDP or NTP to NDPK2 strengthens the interaction significantly between activated phytochrome and NDPK2. To better understand the structural basis of the phytochrome-NDPK2 interaction, we determined the X-ray structures of NDPK1, NDPK2, and dGTP-bound NDPK2 from A.thaliana at 1.8A, 2.6A, and 2.4A, respectively. The structures showed that nucleotide binding caused a slight conformational change in NDPK2 that was confined to helices alphaA and alpha2. This suggests that the presence of nucleotide in the active site and/or the evoked conformational change contributes to the recognition of NDPK2 by activated phytochrome. In vitro binding assays showed that only NDPK2 interacted specifically with the phytochrome and the C-terminal regulatory domain of phytochrome is involved in the interaction. A domain swap experiment between NDPK1 and NDPK2 showed that the variable C-terminal region of NDPK2 is important for the activation by phytochrome. The structure of Arabidopsis NDPK1 and NDPK2 showed that the isoforms share common electrostatic surfaces at the nucleotide-binding site, but the variable C-terminal regions have distinct electrostatic charge distributions. These findings suggest that the binding of nucleotide to NDPK2 plays a regulatory role in phytochrome signaling and that the C-terminal extension of NDPK2 provides a potential binding surface for the specific interaction with phytochromes.

Amino Acid Sequence↗

PubMed2004

Phytochrome phosphorylation modulates light signaling by influencing the protein-protein interaction.

Plant photoreceptor phytochromes are phosphoproteins, but the question as to the functional role of phytochrome phosphorylation has remained to be elucidated. We investigated the functional role of phytochrome phosphorylation in plant light signaling using a Pfr-specific phosphorylation site mutant, Ser598Ala of oat (Avena sativa) phytochrome A (phyA). The transgenic Arabidopsis thaliana (phyA-201 background) plants with this mutant phyA showed hypersensitivity to light, suggesting that phytochrome phosphorylation at Serine-598 (Ser598) in the hinge region is involved in an inhibitory mechanism. The phosphorylation at Ser598 prevented its interaction with putative signal transducers, Nucleoside Diphosphate Kinase-2 and Phytochrome-Interacting Factor-3. These results suggest that phosphorylation in the hinge region of phytochromes serves as a signal-modulating site through the protein-protein interaction between phytochrome and its putative signal transducer proteins.

Avena↗

PubMed2004

9-Hydroxypheophorbide alpha-induced apoptotic death of MCF-7 breast cancer cells is mediated by c-Jun N-terminal kinase activation.

Recently, we synthesized 9-hydroxypheophorbide alpha (9-HPbD), a new chlorophyll-derived photosensitizer. The photo-treatment of MCF-7 human breast cancer cells with 20 kJ/m2 of red light after 5 microM 9-HPbD pretreatment induced cell death, showed typical apoptotic features, i.e., chromatin condensation, phosphatidyl serine externalization, membrane blebbing, and apoptotic bodies with an intact plasma membrane structure. To elucidate the mechanism of 9-HPbD-induced apoptosis, various mediators of the apoptosis were investigated. Release of cytochrome c from mitochondria into the cytosol was distinct 9 h after irradiation, while the levels of most apoptosis-related molecules such as Fas, FasL, Bcl-2, Bax and p53 were unchanged. Furthermore, caspase-9 activated by released cytochrome c was not significantly activated after 9-HPbD-photosensitization. On the other hand, stress-activated protein kinases such as p38 and c-Jun N-terminal kinase (JNK) were activated 1 h after irradiation. Blocking of JNK signaling by transfecting with the dominant negative from of the JNK gene significantly reduced 9-HPbD-induced cell death. Our data show that photosensitization with the new photosensitizer 9-HPbD could induce the apoptotic death of MCF-7 breast cancer cell and that this death is mediated by stress-activated signal through JNK.

Apoptosis↗

PubMed2003

Overexpression of a mutant basic helix-loop-helix protein HFR1, HFR1-deltaN105, activates a branch pathway of light signaling in Arabidopsis.

The HFR1, a basic helix-loop-helix protein, is required for a subset of phytochrome A-mediated photoresponses in Arabidopsis. Here, we show that overexpression of the HFR1-deltaN105 mutant, which lacks the N-terminal 105 amino acids, confers exaggerated photoresponses even in darkness. Physiological analysis implied that overexpression of HFR1-deltaN105 activated constitutively a branch pathway of light signaling that mediates a subset of photomorphogenic responses, including germination, de-etiolation, gravitropic hypocotyl growth, blocking of greening, and expression of some light-regulated genes such as CAB, DRT112, PSAE, PSBL, PORA, and XTR7, without affecting the light-responsiveness of anthocyanin accumulation and expression of other light-regulated genes such as CHS and PSBS. Although the end-of-day far-red light response and petiole elongation were suppressed in the HFR1-deltaN105-overexpressing plants, flowering time was not affected by HFR1-deltaN105. In addition, the HFR1-deltaN105-overexpressing plants showed hypersensitive photoresponses in the inhibition of hypocotyl elongation, dependently on phytochrome A, FHY1, and FHY3 under FR light or phyB under R light, respectively. Moreover, our double mutant analysis suggested that the hypersensitive photoresponse is due to functional cooperation between HFR1-deltaN105 and other light-signaling components including HY5, a basic leucine zipper protein. Taken together, our results of gain-of-function approach with HFR1-deltaN105 suggest the existence of a complex and important basic helix-loop-helix protein-mediated transcriptional network controlling a branch pathway of light signaling and provide a useful framework for further genetic dissection of light-signaling network in Arabidopsis.

Arabidopsis↗

PubMed2003

Functional characterization of phytochrome interacting factor 3 in phytochrome-mediated light signal transduction.

Phytochromes regulate various light responses through their interactions with different signaling proteins, such as phytochrome interacting factor 3 (PIF3). However, the physiological functions of PIF3 in light signaling are not yet fully understood. To increase our understanding of these roles, we characterized a T-DNA insertional pif3 mutant and transgenic plants overexpressing the full-length PIF3. Transgenic overexpressing lines displayed longer hypocotyls and smaller cotyledons under red light and reduced cotyledon opening under both red and far-red light, whereas the pif3 mutant showed the opposite phenotypes. The accumulation of anthocyanin and chlorophyll further indicated complicated features of PIF3 function. The accumulation of anthocyanin was increased and the content of chlorophyll was decreased in the overexpression lines. Our data indicate that PIF3 plays complex roles depending on the type of light response and the light conditions.

Arabidopsis↗

PubMed2003

Production of herbicide-tolerant zoysiagrass by Agrobacterium-mediated transformation.

Herbicide-resistant zoysiagrass (Zoysia japonica Steud.) has been developed by Agrobacterium-mediated transformation. A callus-type transformation system was established by optimizing several factors that affect the rate of transformation, including co-cultivation period and concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), CaCl2 and acetosyringone. Maximal GUS expression was observed when a Type 3 callus was co-cultivated on 2,4-D-free co-cultivation medium for 9 d. In addition, removal of calcium and addition of 50-100 mg/L acetosyringone during co-cultivation enhanced GUS expression. When this optimized protocol was applied to the transformation of the bialaphos resistance gene (bar), four plants per 700 mg of infected calluses survived on the selective medium. DNA gel-blot analysis showed that two copies of the transgene had been integrated. After application of 2 g/L bialaphos for a week the transgenic plants survived herbicide spraying, while untransformed zoysiagrasses and invading weeds died. The herbicide-tolerant zoysiagrass will permit more efficient weed control in this widely cultivated turf grass.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2002

Phytochrome-mediated signal transduction pathways in plants.

Phytochromes are photoreceptors that regulate plant growth and development in response to the solar radiation environment. Recent studies reveal how phytochrome-mediated light signals can be transduced to the cells for their responses. The possible signal transduction pathways of phytochromes include: (a) direct regulation of gene transcription and (b) typical kinase-involved signaling pathways and its regulation by phosphorylation, dephosphorylation, and proteolytic degradation. This review highlights some of the recent findings.

Phytochrome↗

PubMed2002

AtMYB21, a gene encoding a flower-specific transcription factor, is regulated by COP1.

Light is an important environmental signal that governs plant growth and development. One important light-signalling component involved in plant light responses is COP1. The pleiotropic phenotypes of the cop1 mutant suggest that COP1 regulates not only photomorphogenesis, but also other developmental processes. We investigated the role of COP1 by identifying genes that are regulated by COP1. We report that AtMYB21, a gene encoding a flower-specific transcription factor, is ectopically expressed in the cop1 mutant. Analysis shows that dark-grown transgenic seedlings expressing AtMYB21-GR fusion protein display some features of the cop1 mutant, including decreased hypocotyl cell expansion, open cotyledons in the dark, and seedling lethality in the presence of dexamethasone. Light-grown adult transgenic plants expressing AtMYB21 have shorter stems, smaller and narrower leaves, narrower petals, and malformed carpels. In addition, we show that AtMYB21 directly regulates two genes that are also expressed more abundantly in the cop1 mutant. The results indicate that COP1 is required to repress the AtMYB21 gene in seedlings, and the pleiotropic phenotypes shown in the cop1 mutant are due to the combination of misregulation of genuine light-signalling components and other tissue-specific factors.

Arabidopsis↗

PubMed2002

HFR1, a phytochrome A-signalling component, acts in a separate pathway from HY5, downstream of COP1 in Arabidopsis thaliana.

HFR1, a basic helix-loop-helix protein, is known to be required for a subset of phytochrome A (phyA)-dependent photoresponses. To investigate the role of HFR1 in light signalling, we have examined the genetic interaction between HFR1 and HY5, a positive regulator of light signalling, and COP1, a repressor of photomorphogenesis. Double mutant analysis suggests that HFR1 mediates phyA-dependent inhibition of hypocotyl elongation independently of HY5. HFR1 was shown to be necessary for a subset of cop1-triggered photomorphogenic phenotypes in the dark, including inhibition of hypocotyl elongation, gravitropic hypocotyl growth, and expression of the light-inducible genes CAB and RBCS. Phenotypic analysis of the triple mutant cop1hy5hfr1 indicated that both HFR1 and HY5 are required for cop1-mediated photomorphogenic seedling development in darkness, consistent with their additive roles in phyA-dependent signalling. Taken together, these results suggest that HFR1 might act downstream of COP1, in a separate pathway from HY5, to mediate photomorphogenesis in Arabidopsis.

Arabidopsis↗

PubMed2002

A phytochrome-associated protein phosphatase 2A modulates light signals in flowering time control in Arabidopsis.

Reversible protein phosphorylation, which is catalyzed by functionally coupled protein kinases and protein phosphatases, is a major signaling mechanism in eukaryotic cellular functions. The red and far-red light-absorbing phytochrome photoreceptors are light-regulated Ser/Thr-specific protein kinases that regulate diverse photomorphogenic processes in plants. Here, we demonstrate that the phytochromes functionally interact with the catalytic subunit of a Ser/Thr-specific protein phosphatase 2A designated FyPP. The interactions were influenced by phosphorylation status and spectral conformation of the phytochromes. Recombinant FyPP efficiently dephosphorylated oat phytochrome A in the presence of Fe(2+) or Zn(2+) in a spectral form-dependent manner. FyPP was expressed predominantly in floral organs. Transgenic Arabidopsis plants with overexpressed or suppressed FyPP levels exhibited delayed or accelerated flowering, respectively, indicating that FyPP modulates phytochrome-mediated light signals in the timing of flowering. Accordingly, expression patterns of the clock genes in the long-day flowering pathway were altered greatly. These results indicate that a self-regulatory phytochrome kinase-phosphatase coupling is a key signaling component in the photoperiodic control of flowering.

Amino Acid Sequence↗

PubMed2002

Crystal structure of a cyanobacterial phytochrome response regulator.

The two-component signal transduction pathway widespread in prokaryotes, fungi, molds, and some plants involves an elaborate phosphorelay cascade. Rcp1 is the phosphate receiver module in a two-component system controlling the light response of cyanobacteria Synechocystis sp. via cyanobacterial phytochrome Cph1, which recognizes Rcp1 and transfers its phosphoryl group to an aspartate residue in response to light. Here we describe the crystal structure of Rcp1 refined to a crystallographic R-factor of 18.8% at a resolution of 1.9 A. The structure reveals a tightly associated homodimer with monomers comprised of doubly wound five-stranded parallel beta-sheets forming a single-domain protein homologous with the N-terminal activator domain of other response regulators (e.g., chemotaxis protein CheY). The three-dimensional structure of Rcp1 appears consistent with the conserved activation mechanism of phosphate receiver proteins, although in this case, the C-terminal half of its regulatory domain, which undergoes structural changes upon phosphorylation, contributes to the dimerization interface. The involvement of the residues undergoing phosphorylation-induced conformational changes at the dimeric interface suggests that dimerization of Rcp1 may be regulated by phosphorylation, which could affect the interaction of Rcp1 with downstream target molecules.

Amino Acid Sequence↗