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1.
Plant J ; 111(2): 595-607, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35510416

RESUMO

Arabidopsis possesses approximately 2000 transcription factors (TFs) in its genome. They play pivotal roles in various biological processes but analysis of their function has been hampered by the overlapping nature of their activities. To uncover clues to their function, we generated inducible TF lines using glucocorticoid receptor (GR) fusion techniques in Arabidopsis. These TF-GR lines each express one of 1255 TFs as a fusion with the GR gene. An average 14 lines of T2 transgenic TF-GR lines were generated for each TF to monitor their function. To evaluate these transcription lines, we induced the TF-GR lines of phytochrome-interacting factor 4, which controls photomorphogenesis, with synthetic glucocorticoid dexamethasone. These phytochrome-interacting factor 4-GR lines showed the phenotype described in a previous report. We performed screening of the other TF-GR lines for TFs involved in light signaling under blue and far-red light conditions and identified 13 novel TF candidates. Among these, we found two lines showing higher anthocyanin accumulation under light conditions and we examined the regulating genes. These results indicate that the TF-GR lines can be used to dissect functionally redundant genes in plants and demonstrate that the TF-GR line collection can be used as an effective tool for functional analysis of TFs.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fitocromo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Fitocromo/genética , Plantas Geneticamente Modificadas/metabolismo , Receptores de Glucocorticoides/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Spinal Cord ; 60(4): 301-305, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34556821

RESUMO

STUDY DESIGN: A prospective observational study. OBJECTIVES: To depict morphological and functional changes in the cervical nerve roots before and after spinal cord decompression surgery for degenerative cervical myelopathy (DCM). SETTING: A general hospital in Japan. METHODS: Thirteen DCM patients who underwent posterior spinal cord decompression surgery, laminoplasty or laminectomy, were included in this study. The neural foramen shown on MRI and the cross-sectional area (CSA) of the nerve roots on ultrasound were used to evaluate the C5 and C6 nerve roots. The compound muscle action potentials (CMAPs) of deltoid and biceps muscle were also recorded. RESULTS: All patients showed sensorimotor functional improvement without the postoperative C5 palsy after surgery. Foraminal stenosis and preoperative CSA of the nerve root: C4/5 foramen and C5 nerve root, C5/6 foramen and C6 nerve root, had no significant correlation (P = 0.53 and 0.08). CSA of the C5 nerve root displayed no significant change before and after surgery (P = 0.2), however, that of the C6 nerve root reduced significantly after surgery (P = 0.038). The amplitude of the deltoid and biceps CMAPs displayed no significant change before and after surgery (P = 0.05 and 0.05). CONCLUSION: The C6 nerve root CSA change was observed after spinal cord decompression surgery with functional recovery. However, deltoid and biceps CMAPs amplitude showed no significant change. Independent CSA changes on ultrasound might be useful when conducting a functional evaluation of the postoperative nerve root. SPONSORSHIP: The Grant of Japan Orthopaedics and Traumatology Research Foundation No. 395.


Assuntos
Laminoplastia , Doenças da Medula Espinal , Traumatismos da Medula Espinal , Vértebras Cervicais/diagnóstico por imagem , Vértebras Cervicais/cirurgia , Descompressão Cirúrgica , Eletrofisiologia , Humanos , Laminectomia , Paralisia , Doenças da Medula Espinal/cirurgia , Raízes Nervosas Espinhais/diagnóstico por imagem , Raízes Nervosas Espinhais/cirurgia
3.
J Cell Sci ; 131(16)2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-30030372

RESUMO

Stress granules, a type of cytoplasmic RNA granule in eukaryotic cells, are induced in response to various environmental stresses, including high temperature. However, how high temperatures induce the formation of these stress granules in plant cells is largely unknown. Here, we characterized the process of stress granule formation in Arabidopsis thaliana by combining live imaging and electron microscopy analysis. In seedlings grown at 22°C, stress granule formation was induced at temperatures above a critical threshold level of 34°C in the absence of transpiration. The threshold temperature was the same, regardless of whether the seedlings were grown at 22°C or 4°C. High-resolution live imaging microscopy revealed that stress granule formation is not correlated with the sizes of pre-existing RNA processing bodies (P-bodies) but that the two structures often associated rapidly. Immunoelectron microscopy revealed a previously unidentified characteristic of the fine structures of Arabidopsis stress granules and P-bodies: the lack of ribosomes and the presence of characteristic electron-dense globular and filamentous structures. These results provide new insights into the universal nature of stress granules in eukaryotic cells.


Assuntos
Arabidopsis/fisiologia , Grânulos Citoplasmáticos/metabolismo , Temperatura Alta , Estresse Fisiológico/fisiologia , Temperatura , Arabidopsis/genética , Arabidopsis/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Resposta ao Choque Térmico/fisiologia , Plantas Geneticamente Modificadas , Plântula
5.
BMC Nephrol ; 20(1): 470, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852449

RESUMO

BACKGROUND: A simpler method for detecting atherosclerosis obliterans is required in the clinical setting. Laser Doppler flowmetry (LDF) is easy to perform and can accurately detect deterioration in skin perfusion. We performed LDF for hemodialysis patients to determine the correlations between blood flow in the lower limbs and peripheral arterial disease (PAD). METHODS: This retrospective study included 128 hemodialysis patients. Patients were categorized into the non-PAD group (n = 106) and PAD group (n = 22), 14 early stage PAD patients were included in the PAD group. We conducted LDF for the plantar area and dorsal area of the foot and examined skin perfusion pressure (SPP) during dialysis. RESULTS: SPP-Dorsal Area values were 82.1 ± 22.0 mmHg in the non-PAD, and 59.1 ± 20.3 mmHg in PAD group, respectively (p < 0.05). The LDF-Plantar blood flow (Qb) values were 32.7 ± 15.5 mL/min in non-PAD group and 21.5 ± 11.3 mL/min in PAD group (p < 0.001). A total of 21 non-PAD patients underwent LDF before and during dialysis. The LDF-Plantar-Qb values were 36.5 ± 17.6 mL/min before dialysis and 29.6 ± 17.7 mL/min after dialysis (p < 0.05). We adjusted SPP and LDF for PAD using logistic regression, SPP-Dorsal-Area and LDF-P were significantly correlated with PAD (p < 0.05). The receiver-operating characteristic curve analysis indicated cut-off values of 20.0 mL/min for LDF-Plantar-Qb during dialysis. CONCLUSION: LDF is a simple technique for sensitive detection of early-stage PAD. This assessment will help physicians identify early-stage PAD, including Fontaine stage II in clinical practice, thereby allowing prompt treatment.


Assuntos
Fluxometria por Laser-Doppler/métodos , Extremidade Inferior/irrigação sanguínea , Doença Arterial Periférica/diagnóstico , Doença Arterial Periférica/etiologia , Diálise Renal/efeitos adversos , Idoso , Idoso de 80 Anos ou mais , Diagnóstico Precoce , Feminino , Humanos , Falência Renal Crônica/diagnóstico , Falência Renal Crônica/fisiopatologia , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/fisiopatologia , Estudos Retrospectivos
6.
Inorg Chem ; 57(18): 11322-11332, 2018 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-30168718

RESUMO

Novel dinuclear gold(I)- and copper(I)- N-heterocyclic carbene (NHC, L-1a) complexes [M2(L-1a)](PF6)2 (M = AuI (Au-1·PF6) and M = CuI (Cu-1·PF6)) were synthesized by transmetalation of the dinuclear silver(I)-NHC complex [Ag2(L-1a)](PF6)2 (Ag-1·PF6) with [AuCl(Me2S)] or CuI in over 70% yield. These NHC complexes were characterized by CHN elemental analysis, Fourier transform infrared spectroscopy, thermogravimetry/differential thermal analysis, and solution (1H and 13C) NMR spectroscopy. X-ray crystallography revealed that Au-1·PF6 and Cu-1·PF6 are dinuclear molecules consisting of two linear intramolecular C-M-C bonds (M = AuI and CuI), one M···M interaction, and two metal atoms arranged in a T-shaped geometry; their molecular structures are very similar to the basket-shaped cage structure of the parent complex Ag-1·PF6. Because of the smaller ionic radius of copper(I), Cu-1·PF6 has the smallest upper space of the basket among the three complexes. Au-1·PF6 and Cu-1·PF6 were very stable in the solid state and in solution. They did not undergo nucleophilic reaction with thiols 2-mercaptoethanol and 2-benzimidazolethiol (Hbmt) at least for several hours, whereas Ag-1·PF6 did react, forming precipitates of silver(I) thiolate and the free ligand L-1a·PF6. The minimum inhibitory concentration values toward a panel of bacteria, yeasts, and molds were examined in a water-suspension system and a solution system containing dimethyl sulfoxide. The antimicrobial spectra of the three compounds were metal-dependent, and Au-1·PF6 showed the greatest activity toward Gram-positive bacteria.


Assuntos
Cobre/química , Cobre/farmacologia , Ouro/química , Ouro/farmacologia , Metano/análogos & derivados , Microbiota/efeitos dos fármacos , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Cristalografia por Raios X , Fungos/efeitos dos fármacos , Metano/química , Metano/farmacologia
7.
Biochem Biophys Res Commun ; 485(1): 174-180, 2017 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-28189687

RESUMO

14-3-3 proteins regulate numerous cellular processes through interaction with their target proteins in a phosphorylation dependent manner. Although proteins that are regulated by 14-3-3s have been studied, the regulatory mechanism of 14-3-3s is poorly understood. In the present study, F-box proteins, a component of Skp1-Cullin-F-box E3 ubiquitin ligase, were identified as 14-3-3 targets using yeast two-hybrid screening. Among them, AtSKIP18 and AtSKIP31, were shown to mediate the degradation of Arabidopsis 14-3-3s. Mutational analyses of AtSKIP18 and AtSKIP31 indicated that the phosphorylation of AtSKIPs is critical for interaction and degradation of 14-3-3s. The loss-of-function mutation in AtSKIP31 resulted in enhanced primary root growth under nitrogen deficient conditions. These findings suggest that AtSKIP31 regulates the primary root growth in nitrogen deficiency via degrading 14-3-3s.


Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas F-Box/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Arabidopsis/crescimento & desenvolvimento , Fosforilação , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Mapas de Interação de Proteínas , Proteólise , Ubiquitinação
8.
Plant Cell ; 26(2): 712-28, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24563205

RESUMO

CULLIN4-RING E3 ubiquitin ligases (CRL4s) regulate key developmental and stress responses in eukaryotes. Studies in both animals and plants have led to the identification of many CRL4 targets as well as specific regulatory mechanisms that modulate their function. The latter involve COP10-DET1-DDB1 (CDD)-related complexes, which have been proposed to facilitate target recognition by CRL4, although the molecular basis for this activity remains largely unknown. Here, we provide evidence that Arabidopsis thaliana DET1-, DDB1-ASSOCIATED1 (DDA1), as part of the CDD complex, provides substrate specificity for CRL4 by interacting with ubiquitination targets. Thus, we show that DDA1 binds to the abscisic acid (ABA) receptor PYL8, as well as PYL4 and PYL9, in vivo and facilitates its proteasomal degradation. Accordingly, we found that DDA1 negatively regulates ABA-mediated developmental responses, including inhibition of seed germination, seedling establishment, and root growth. All other CDD components displayed a similar regulatory function, although they did not directly interact with PYL8. Interestingly, DDA1-mediated destabilization of PYL8 is counteracted by ABA, which protects PYL8 by limiting its polyubiquitination. Altogether, our data establish a function for DDA1 as a substrate receptor for CRL4-CDD complexes and uncover a mechanism for the desensitization of ABA signaling based on the regulation of ABA receptor stability.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteólise , Receptores de Superfície Celular/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Mutação/genética , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Ubiquitinação
9.
J Exp Bot ; 67(11): 3471-9, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27126796

RESUMO

The mitogen-activated protein kinases (MAPKs/MPKs) are important factors in the regulation of signal transduction in response to biotic and abiotic stresses. Previously, we characterized a MAPK from tobacco, Nicotiana tabacum MPK4 (NtMPK4). Here, we found a highly homologous gene, NtMPK4-like (NtMPK4L), in tobacco as well as other species in Solanaceae and Gramineae. Deduced amino acid sequences of their translation products carried MEY motifs instead of conserved TXY motifs of the MAPK family. We isolated the full length NtMPK4L gene and examined the physiological functions of NtMPK4L. We revealed that NtMPK4L was activated by wounding, like NtMPK4. However, a constitutively active salicylic acid-induced protein kinase kinase (SIPKK(EE)), which phosphorylates NtMPK4, did not phosphorylate NtMPK4L. Moreover, a tyrosine residue in the MEY motif was not involved in NtMPK4L activation. We also found that NtMPK4L-silenced plants showed rapid transpiration caused by remarkably open stomata. In addition, NtMPK4L-silenced plants completely lost the ability to close stomata upon ozone treatment and were highly sensitive to ozone, suggesting that this atypical MAPK plays a role in ozone tolerance through stomatal regulation.


Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas Quinases Ativadas por Mitógeno/genética , Nicotiana/genética , Ozônio/metabolismo , Proteínas de Plantas/genética , Estômatos de Plantas/metabolismo , Sequência de Aminoácidos , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Nicotiana/enzimologia , Nicotiana/metabolismo
10.
J Proteome Res ; 13(7): 3223-30, 2014 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-24846764

RESUMO

The 26S proteasome is an ATP-dependent proteinase complex that is responsible for regulated proteolysis of polyubiquitinated proteins in eukaryotic cells. Here, we report novel 26S proteasome interacting proteins in Arabidopsis as revealed by LC-MS/MS analysis. We performed a two-step screening process that involved affinity purification of the 26S proteasome using Arabidopsis plants expressing a FLAG-tagged RPT2a subunit and partial purification of the 26S proteasome from cultured cells by glycerol density gradient centrifugation (GDG). Two plastid proteins, LTA2 and PDH E1α, which were commonly identified by both affinity purification and GDG, interacted with the 26S proteasome both in vitro and in vivo, and the transit peptides of LTA2 and PDH E1α were necessary for the interaction. Furthermore, the degradation of both LTA2 and PDH E1α was inhibited by MG132, a proteasome inhibitor. Similar to those two proteins, 26S proteasome subunits RPT2a/b and RPT5a interacted with the transit peptides of three other chloroplast proteins, which are known to be substrates of the ubiquitin-26S proteasome system. These results suggest that a direct interaction between the 26S proteasome and a transit peptide is important for the degradation of unimported plastid protein precursors to maintain cellular homeostasis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Fragmentos de Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Precursores de Proteínas/metabolismo , Proteólise , Arabidopsis/metabolismo , Mapeamento de Interação de Proteínas , Proteômica , Plântula/metabolismo , Espectrometria de Massas em Tandem
11.
Plant Cell ; 22(1): 108-23, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20061554

RESUMO

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) possesses E3 ligase activity and promotes degradation of key factors involved in the light regulation of plant development. The finding that CULLIN4 (CUL4)-Damaged DNA Binding Protein1 (DDB1) interacts with DDB1 binding WD40 (DWD) proteins to act as E3 ligases implied that CUL4-DDB1 may associate with COP1-SUPPRESSOR OF PHYA (SPA) protein complexes, since COP1 and SPAs are DWD proteins. Here, we demonstrate that CUL4-DDB1 physically associates with COP1-SPA complexes in vitro and in vivo, likely via direct interaction of DDB1 with COP1 and SPAs. The interactions between DDB1 and COP1, SPA1, and SPA3 were disrupted by mutations in the WDXR motifs of MBP-COP1, His-SPA1, and His-SPA3. CUL4 cosuppression mutants enhanced weak cop1 photomorphogenesis and flowered early under short days. Early flowering of short day-grown cul4 mutants correlated with increased FLOWERING LOCUS T transcript levels, whereas CONSTANS transcript levels were not altered. De-etiolated1 and COP1 can bind DDB1 and may work with CUL4-DDB1 in distinct complexes, but they mediate photomorphogenesis in concert. Thus, a series of CUL4-DDB1-COP1-SPA E3 ligase complexes may mediate the repression of photomorphogenesis and, possibly, of flowering time.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ligação a DNA/metabolismo , Flores/crescimento & desenvolvimento , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/genética , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Luz , Dados de Sequência Molecular , Mutação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , RNA de Plantas/genética , Ubiquitina-Proteína Ligases/genética
12.
J Plant Res ; 126(3): 387-94, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23076439

RESUMO

Angiosperms have a unique sexual reproduction system called "double fertilization." One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.


Assuntos
Arabidopsis/fisiologia , Membrana Celular/fisiologia , Fertilização , Células Germinativas Vegetais/fisiologia , Arabidopsis/citologia , Arabidopsis/genética , Células Germinativas Vegetais/citologia , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Imagem com Lapso de Tempo
13.
PLoS One ; 18(2): e0281767, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36795787

RESUMO

Previously, we developed a technique to introduce a superfolder green fluorescent protein (sGFP) fusion protein directly into plant cells using atmospheric-pressure plasma. In this study, we attempted genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system using this protein introduction technique. As an experimental system to evaluate genome editing, we utilized transgenic reporter plants carrying the reporter genes L-(I-SceI)-UC and sGFP-waxy-HPT. The L-(I-SceI)-UC system allowed the detection of successful genome editing by measuring the chemiluminescent signal observed upon re-functionalization of the luciferase (LUC) gene following genome editing. Similarly, the sGFP-waxy-HPT system conferred hygromycin resistance caused by hygromycin phosphotransferase (HPT) during genome editing. CRISPR/Cas9 ribonucleoproteins targeting these reporter genes were directly introduced into rice calli or tobacco leaf pieces after treatment with N2 and/or CO2 plasma. Cultivation of the treated rice calli on a suitable medium plate produced the luminescence signal, which was not observed in the negative control. Four types of genome-edited sequences were obtained upon sequencing the reporter genes of genome-edited candidate calli. sGFP-waxy-HPT-carrying tobacco cells exhibited hygromycin resistance during genome editing. After repeated cultivation of the treated tobacco leaf pieces on a regeneration medium plate, the calli were observed with leaf pieces. A green callus that was hygromycin-resistant was harvested, and a genome-edited sequence in the tobacco reporter gene was confirmed. As direct introduction of the Cas9/sgRNA (single guide RNA) complex using plasma enables genome editing in plants without any DNA introduction, this method is expected to be optimized for many plant species and may be widely applied for plant breeding in the future.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Células Vegetais , Temperatura , Melhoramento Vegetal , Plantas Geneticamente Modificadas/genética , Genoma de Planta
14.
J Proteome Res ; 11(1): 68-78, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22029422

RESUMO

Proteomics is a useful analytical approach for investigating crop responses to stress. Recent remarkable advances in proteomic techniques allow for the identification of a wider range of proteins than was previously possible. The application of proteomic techniques to clarify the molecular mechanisms underlying crop responses to flooding stress may facilitate the development of flood tolerant crops. Flooding is an environmental stress found worldwide and may increase in frequency due to changes in global climate. Waterlogging resulting from flooding causes significant reductions in the growth and yield of several crops. Transient flooding displaces gases in soil pores and often causes hypoxia in plants grown on land with poor drainage. Changes in protein expression and post-translational modification of proteins occur as plants activate their defense system in response to flooding stress. In this review, we discuss the contributions that proteomic studies have made toward increasing our understanding of the well-organized cellular response to flooding in soybean and other crops. The biological relevance of the proteins identified using proteomic techniques in regard to crop stress tolerance will be discussed as well.


Assuntos
Produtos Agrícolas/fisiologia , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Estresse Fisiológico , Produtos Agrícolas/citologia , Produtos Agrícolas/metabolismo , Inundações , Organelas/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica , Glycine max/citologia , Glycine max/metabolismo , Glycine max/fisiologia
15.
Plant Biotechnol (Tokyo) ; 39(2): 179-183, 2022 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-35937533

RESUMO

Previously, we developed a method that uses temperature-controlled atmospheric-pressure plasma to induce protein uptake in plant cells. In the present work, we examined the mechanism underlying such uptake of a fluorescent-tagged protein in tobacco leaf cells. Intact leaf tissue was irradiated with N2 plasma generated by a multi-gas plasma jet and then exposed to the test protein (histidine-tagged superfolder green fluorescence protein fused to adenylate cyclase); fluorescence intensity was then monitored over time as an index of protein uptake. Confocal microscopy revealed that protein uptake potential was retained in the leaf tissue for at least 3 h after plasma treatment. Further examination indicated that the introduced protein reached a similar amount to that after overnight incubation at approximately 5 h after irradiation. Inhibitor experiments revealed that protein uptake was significantly suppressed compared with negative controls by pretreatment with sodium azide (inhibitor of adenosine triphosphate hydrolysis) or sucrose or brefeldin A (inhibitors of clathrin-mediated endocytosis) but not by pretreatment with genistein (inhibitor of caveolae/raft-mediated endocytosis) or cytochalasin D (inhibitor of micropinocytosis/phagocytosis), indicating that the N2 plasma treatment induced protein transportation across the plant plasma membrane via clathrin-mediated endocytosis.

16.
Case Rep Orthop ; 2021: 5544126, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33976947

RESUMO

Pyogenic facet joint infection (PFJI) is a relatively rare spinal infection. Clinical suspicion of this condition is a key for diagnosis. We report a case of PFJI which required decompression surgery for severe neurological dysfunction. The patient was a 44-year-old woman who had a previous history of orthotic therapy for idiopathic scoliosis. The patient was admitted to our hospital with a history of two days of high fever and severe low back pain. There was no neurologic deficit, and blood tests revealed high levels of inflammatory markers. There was a slight amount of fluid that had collected at L4/5 facet joint in lumbar MRI. She was admitted for examination and treatment of fever of unknown origin and low back pain. Antibiotic treatment started the day after hospitalization since the first report of the blood culture taken upon admission tested positive to gram-positive cocci. As low back pain and fever persisted, an MRI was taken again on the fifth day of hospitalization. Repeated MRI showed fluid extension from the left facet joint to paravertebral muscles and epidural space. She was diagnosed with PFJI, and facet joint puncture was performed. At this time, it became clear that she had foot drop on the right, the contralateral side of the PFJI. She underwent irrigation, debridement, and partial laminectomy. Methicillin-sensitive Staphylococcus aureus (MSSA) was detected in blood cultures at the time of hospitalization, in the puncture fluid and tissue collected during surgery. The patient recovered completely from foot drop after the operation and a three-month course of antibiotics. As the imaging findings may be inadequate in the early stages of onset and PFJI potentially causes neurologic deficit such as foot drop, neurological findings need to be carefully observed even after hospitalization and one should reexamine the MRI if symptoms or clinical findings did not improve or were aggravated.

17.
BMC Plant Biol ; 10: 200, 2010 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-20836890

RESUMO

BACKGROUND: Phosphoenolpyruvate carboxylase (PEPC) is a critical enzyme catalyzing the ß-carboxylation of phosphoenolpyruvate (PEP) to oxaloacetate, a tricarboxylic acid (TCA) cycle intermediate. PEPC typically exists as a Class-1 PEPC homotetramer composed of plant-type PEPC (PTPC) polypeptides, and two of the subunits were reported to be monoubiquitinated in germinating castor oil seeds. By the large-scale purification of ubiquitin (Ub)-related proteins from lily anther, two types of PEPCs, bacterial-type PEPC (BTPC) and plant-type PEPC (PTPC), were identified in our study as candidate Ub-related proteins. Until now, there has been no information about the properties of the PEPCs expressed in male reproductive tissues of higher plants. RESULTS: Expression analyses showed that lily BTPC (LlBTPC) and Arabidopsis BTPC (AtBTPC) were significantly expressed in pollen. The fusion protein AtBTPC-Venus localized in the cytoplasm of the vegetative cell (VC). Both LlBTPC and AtBTPC expression initiated after the last mitosis before pollen germination. Lily PTPC (LlPTPC) and monoubiquitinated LlPTPC (Ub-LlPTPC) remained at constant levels during pollen development. In late bicellular pollen of lily, LlBTPC forms a hetero-octameric Class-2 PEPC complex with LlPTPC to express PEPC activity. CONCLUSION: Our results suggest that an LlBTPC:Ub-LlPTPC:LlPTPC complex is formed in the VC cytoplasm during late pollen development. Both LlBTPC and AtBTPC expression patterns are similar to the patterns of the appearance of storage organelles during pollen development in lily and Arabidopsis, respectively. Therefore, BTPC is thought to accelerate the metabolic flow for the synthesis of storage substances during pollen maturation. Our study provides the first characterization of BTPC in pollen, the male gametophyte of higher plants.


Assuntos
Arabidopsis/enzimologia , Lilium/enzimologia , Fosfoenolpiruvato Carboxilase/metabolismo , Pólen/enzimologia , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética
18.
Plants (Basel) ; 9(9)2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32825459

RESUMO

Xanthomonas campestris is one of bacteria carrying a type III secretion system which transports their effector proteins into host plant cells to disturb host defense system for their infection. To establish a genome editing system without introducing any foreign gene, we attempted to introduce genome editing enzymes through the type III secretion system. In a test of protein transfer, X. campestris pv. campestris (Xcc) transported a considerable amount of a reporter protein sGFP-CyaA into tobacco plant cells under the control of the type III secretion system while maintaining cell viability. For proof of concept for genome editing, we used a reporter tobacco plant containing a luciferase (LUC) gene interrupted by a meganuclease I-SceI recognition sequence; this plant exhibits chemiluminescence of LUC only when a frameshift mutation is introduced at the I-SceI recognition site. Luciferase signal was observed in tobacco leaves infected by Xcc carrying an I-SceI gene which secretes I-SceI protein through the type III system, but not leaves infected by Xcc carrying a vector control. Genome-edited tobacco plant could be regenerated from a piece of infected leaf piece by repeated selection of LUC positive calli. Sequence analysis revealed that the regenerated tobacco plant possessed a base deletion in the I-SceI recognition sequence that activated the LUC gene, indicating genome editing by I-SceI protein transferred through the type III secretion system of Xcc.

19.
J Exp Bot ; 60(11): 3067-73, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19429840

RESUMO

The majority of proteins in eukaryotic cells are modified according to highly regulated mechanisms to fulfill specific functions and to achieve localization, stability, and transport. Protein ubiquitination is one of the major post-translational modifications occurring in eukaryotic cells. To obtain the proteomic dataset related to the ubiquitin (Ub)-dependent regulatory system in Arabidopsis, affinity purification with an anti-Ub antibody under native condition was performed. Using MS/MS analysis, 196 distinct proteins represented by 251 distinct genes were identified. The identified proteins were involved in metabolism (23.0%), stress response (21.4%), translation (16.8%), transport (6.7%), cell morphology (3.6%), and signal transduction (1.5%), in addition to proteolysis (16.8%) to which proteasome subunits (14.3%) is included. On the basis of potential ubiquitination-targeting signal motifs, in-gel mobilities, and previous reports, 78 of the identified proteins were classified as ubiquitinated proteins and the rest were speculated to be associated proteins of ubiquitinated proteins. The degradation of three proteins predicted to be ubiquitinated proteins was inhibited by a proteasome inhibitor, suggesting that the proteins were regulated by Ub/proteasome-dependent proteolysis.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/isolamento & purificação , Arabidopsis/química , Ubiquitina/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteômica , Plântula/química , Plântula/genética , Plântula/metabolismo , Ubiquitinação
20.
Biosci Biotechnol Biochem ; 73(4): 865-9, 2009 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-19352029

RESUMO

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) of eukaryotic cells triggers the transcriptional induction of ER-resident molecular chaperones to maintain cellular homeostasis, termed the ER stress response. Previously we isolated AtbZIP60, a membrane-bound transcription factor involved in the Arabidopsis ER stress response whose activity is controlled by proteolytic cleavage. In this study we characterized the active form of AtbZIP60 localized in the nucleus during the ER stress response. Transient assay using Arabidopsis protoplasts revealed that activation of BiP promoters by AtbZIP60 is dependent on the cis-elements plant-unfolded protein response element (P-UPRE) and ER stress response element (ERSE). Transcriptional activation activity of AtbZIP60 was mainly located in the region for amino acids 41-80 of AtbZIP60. Size exclusion chromatography analysis showed that the nuclear form of AtbZIP60 exists as a protein complex of approximately 260 kDa. On the basis of the present study combined with observations described in the literature, possible mechanisms of AtbZIP60's action in the nucleus are discussed.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Estresse Fisiológico , Arabidopsis/genética , Arabidopsis/fisiologia , Sequência de Bases , Genes de Plantas/genética , Humanos , Regiões Promotoras Genéticas , Desnaturação Proteica , Elementos de Resposta/genética , Ativação Transcricional
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