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1.
Mol Plant ; 13(3): 446-458, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31899321

RESUMO

Post-translational modifications play essential roles in finely modulating eukaryotic circadian clock systems. In plants, the effects of O-glycosylation on the circadian clock and the underlying mechanisms remain largely unknown. The O-fucosyltransferase SPINDLY (SPY) and the O-GlcNAc transferase SECRET AGENT (SEC) are two prominent O-glycosylation enzymes in higher plants, with both overlapped and unique functions in plant growth and development. Unlike the critical role of O-GlcNAc in regulating the animal circadian clock, here we report that nuclear-localized SPY, but not SEC, specifically modulates the pace of the Arabidopsis circadian clock. By identifying the interactome of SPY, we identified PSEUDO-RESPONSE REGULATOR 5 (PRR5), one of the core circadian clock components, as a new SPY-interacting protein. PRR5 can be O-fucosylated by SPY in planta, while point mutation in the catalytic domain of SPY abolishes the O-fucosylation of PRR5. The protein abundance of PRR5 is strongly increased in spy mutants, while the degradation rate of PRR5 is much reduced, suggesting that PRR5 proteolysis is promoted by SPY-mediated O-fucosylation. Moreover, multiple lines of genetic evidence indicate that PRR5 is a major downstream target of SPY to specifically mediate its modulation of the circadian clock. Collectively, our findings provide novel insights into the specific role of the O-fucosyltransferase activity of SPY in modulating the circadian clock and implicate that O-glycosylation might play an evolutionarily conserved role in modulating the circadian clock system, via O-GlcNAcylation in mammals, but via O-fucosylation in higher plants.

2.
Nat Commun ; 10(1): 2630, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201314

RESUMO

Phytochromes initiate chloroplast biogenesis by activating genes encoding the photosynthetic apparatus, including photosynthesis-associated plastid-encoded genes (PhAPGs). PhAPGs are transcribed by a bacterial-type RNA polymerase (PEP), but how phytochromes in the nucleus activate chloroplast gene expression remains enigmatic. We report here a forward genetic screen in Arabidopsis that identified NUCLEAR CONTROL OF PEP ACTIVITY (NCP) as a necessary component of phytochrome signaling for PhAPG activation. NCP is dual-targeted to plastids and the nucleus. While nuclear NCP mediates the degradation of two repressors of chloroplast biogenesis, PIF1 and PIF3, NCP in plastids promotes the assembly of the PEP complex for PhAPG transcription. NCP and its paralog RCB are non-catalytic thioredoxin-like proteins that diverged in seed plants to adopt nonredundant functions in phytochrome signaling. These results support a model in which phytochromes control PhAPG expression through light-dependent double nuclear and plastidial switches that are linked by evolutionarily conserved and dual-localized regulatory proteins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Chaperonas Moleculares/metabolismo , Fitocromo/metabolismo , Transcrição Genética/fisiologia , Arabidopsis/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cloroplastos/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Fotossíntese/fisiologia , Plantas Geneticamente Modificadas , Plastídeos/genética , Plastídeos/metabolismo , Transdução de Sinais/fisiologia , Transcrição Genética/efeitos da radiação
3.
Anal Chem ; 91(9): 6345-6352, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-30916925

RESUMO

Molecular advances have been made in analysis systems for a wide variety of applications ranging from biodiagnostics, biosafety, bioengineering, and biofuel research applications. There are, however, limited practical tools necessary for in situ and accurate detection of nucleic acid targets during field work. New technology is needed to translate these molecular advances from laboratory settings into the real-life practical monitoring realm. The exquisite characteristics (e.g., sensitivity and adaptability) of plasmonic nanosensors have made them attractive candidates for field-ready sensing applications. Herein, we have developed a fiber-based plasmonic sensor capable of direct detection (i.e., no washing steps required) of nucleic acid targets, which can be detected simply by immerging the sensor in the sample solution. This sensor is composed of an optical fiber that is decorated with plasmonic nanoprobes based on silver-coated gold nanostars (AuNS@Ag) to detect target nucleic acids using the surface-enhanced Raman scattering (SERS) sensing mechanism of nanoprobes referred to as inverse molecular sentinels (iMS). These fiber-optrodes can be reused for several detection-regeneration cycles (>6). The usefulness and applicability of the iMS fiber-sensors was tested by detecting target miRNA in extracts from leaves of plants that were induced to have different expression levels of miRNA targets. These fiber-optrodes enable direct detection of miRNA in plant tissue extract without the need for complex assays by simply immersing the fiber in the sample solution. The results indicate the fiber-based sensors developed herein have the potential to be a powerful tool for field and in situ analysis of nucleic acid samples.

4.
ACS Appl Mater Interfaces ; 11(8): 7743-7754, 2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30694650

RESUMO

Monitoring gene expression within whole plants is critical for many applications ranging from plant biology to agricultural biotechnology and biofuel development; however, no method currently exists for in vivo monitoring of genomic targets in plant systems without requiring sample extraction. Herein, we report a unique multimodal method based on plasmonic nanoprobes capable of in vivo imaging and biosensing of microRNA biotargets within whole plant leaves by integrating three different and complementary techniques: surface-enhanced Raman scattering (SERS), X-ray fluorescence (XRF), and plasmonics-enhanced two-photon luminescence (TPL). The method developed uses plasmonic nanostars, which not only provide large Raman signal enhancement but also allow for localization and quantification by XRF and plasmonics-enhanced TPL, owing to gold content and high two-photon luminescence cross sections. Our method uses inverse molecular sentinel nanoprobes for SERS bioimaging of microRNA within Arabidopsis thaliana leaves to provide a dynamic SERS map of detected microRNA targets while also quantifying nanoprobe concentrations using XRF and TPL. The nanoprobes were observed to occupy the intercellular spaces upon infiltration into the leaf tissues. This report lays the foundation for the use of plasmonic nanoprobes for in vivo functional imaging of nucleic acid biotargets in whole plants, a tool that will revolutionize bioengineering research by allowing the study of these biotargets with previously unmet spatial and temporal resolution, 200 µm and 30 min, respectively.


Assuntos
Arabidopsis/genética , MicroRNAs/metabolismo , Arabidopsis/metabolismo , Técnicas Biossensoriais , Carbocianinas/química , Ouro/química , Nanopartículas Metálicas/química , MicroRNAs/química , Folhas de Planta/genética , Folhas de Planta/metabolismo , Prata/química , Espectrometria por Raios X , Análise Espectral Raman
5.
Sci Rep ; 8(1): 12043, 2018 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-30104574

RESUMO

Parthenocarpy, or pollination-independent fruit set, is an attractive trait for fruit production and can be induced by increased responses to the phytohormone gibberellin (GA), which regulates diverse aspects of plant development. GA signaling in plants is negatively regulated by DELLA proteins. A loss-of-function mutant of tomato DELLA (SlDELLA), procera (pro) thus exhibits enhanced GA-response phenotypes including parthenocarpy, although the pro mutation also confers some disadvantages for practical breeding. This study identified a new milder hypomorphic allele of SlDELLA, procera-2 (pro-2), which showed weaker GA-response phenotypes than pro. The pro-2 mutant contains a single nucleotide substitution, corresponding to a single amino acid substitution in the SAW subdomain of the SlDELLA. Accumulation of the mutated SlDELLA transcripts in wild-type (WT) resulted in parthenocarpy, while introduction of intact SlDELLA into pro-2 rescued mutant phenotypes. Yeast two-hybrid assays revealed that SlDELLA interacted with three tomato homologues of GID1 GA receptors with increasing affinity upon GA treatment, while their interactions were reduced by the pro and pro-2 mutations. Both pro and pro-2 mutants produced higher fruit yields under high temperature conditions, which were resulted from higher fruit set efficiency, demonstrating the potential for genetic parthenocarpy to improve yield under adverse environmental conditions.


Assuntos
Frutas/crescimento & desenvolvimento , Giberelinas/genética , Giberelinas/metabolismo , Lycopersicon esculentum/genética , Proteínas de Plantas/genética , Alelos , Substituição de Aminoácidos/genética , Regulação da Expressão Gênica de Plantas/genética , Reguladores de Crescimento de Planta/genética , Polimorfismo de Nucleotídeo Único/genética , Receptores de Superfície Celular/metabolismo , Triazóis/farmacologia
6.
Plant Cell ; 30(8): 1710-1728, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30008445

RESUMO

Fruit initiation following fertilization in angiosperms is strictly regulated by phytohormones. In tomato (Solanum lycopersicum), auxin and gibberellin (GA) play central roles in promoting fruit initiation. Without fertilization, elevated GA or auxin signaling can induce parthenocarpy (seedless fruit production). The GA-signaling repressor SlDELLA and auxin-signaling components SlIAA9 and SlARF7 repress parthenocarpy, but the underlying mechanism is unknown. Here, we show that SlDELLA and the SlARF7/SlIAA9 complex mediate crosstalk between GA and auxin pathways to regulate fruit initiation. Yeast-two-hybrid and coimmunoprecipitation assays showed that SlARF7 and additional activator SlARFs interact with SlDELLA and SlIAA9 through distinct domains. SlARF7/SlIAA9 and SlDELLA antagonistically modulate the expression of feedback-regulated genes involved in GA and auxin metabolism, whereas SlARF7/SlIAA9 and SlDELLA coregulate the expression of fruit growth-related genes. Analysis of procera (della), SlARF7 RNAi (with downregulated expression of multiple activator SlARFs), and entire (iaa9) single and double mutants indicated that these genes additively affect parthenocarpy, supporting the notion that the SlARFs/SlIAA9 and SlDELLA interaction plays an important role in regulating fruit initiation. Analysis of the GA-deficient mutant gib1 showed that active GA biosynthesis and signaling are required for auxin-induced fruit initiation. Our study reveals how direct crosstalk between auxin- and GA-signaling components is critical for tomato fruit initiation.


Assuntos
Frutas/metabolismo , Giberelinas/metabolismo , Ácidos Indolacéticos/metabolismo , Lycopersicon esculentum/metabolismo , Proteínas de Plantas/metabolismo , Frutas/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Lycopersicon esculentum/genética , Proteínas de Plantas/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
7.
J Healthc Eng ; 2017: 8465212, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29065653

RESUMO

Background. The measurement of the functional range of motion (FROM) of lower limb joints is an essential parameter for gait analysis especially in evaluating rehabilitation programs. Aim. To develop a simple, reliable, and affordable mechanical goniometer (MGR) for gait analysis, with six-degree freedom to dynamically assess lower limb joint angles. Design. Randomized control trials, in which a new MGR was developed for the measurements of FROM of lower limb joints. Setting. Reliability of the designed MGR was evaluated and validated by a motion analysis system (MAS). Population. Thirty healthy subjects participated in this study. Methods. Reliability and validity of the new MGR were tested by intraclass correlation coefficient (ICC), Bland-Altman plots, and linear correlation analysis. Results. The MGR has good inter- and intrarater reliability and validity with ICC ≥ 0.93 (for both). Moreover, measurements made by MGR and MAS were comparable and repeatable with each other, as confirmed by Bland-Altman plots. Furthermore, a very high degree of linear correlation (R ≥ 0.92 for all joint angle measurements) was found between the lower limb joint angles measured by MGR and MAS. Conclusion. A simple, reliable, and affordable MGR has been designed and developed to aid clinical assessment and treatment evaluation of gait disorders.


Assuntos
Articulação do Tornozelo/fisiologia , Artrometria Articular , Transtornos Neurológicos da Marcha/diagnóstico , Articulação do Quadril/fisiologia , Articulação do Joelho/fisiologia , Amplitude de Movimento Articular , Adulto , Fenômenos Biomecânicos , Técnicas de Apoio para a Decisão , Feminino , Marcha , Transtornos Neurológicos da Marcha/fisiopatologia , Voluntários Saudáveis , Humanos , Masculino , Movimento (Física) , Variações Dependentes do Observador , Análise de Regressão , Reprodutibilidade dos Testes , Estresse Mecânico , Adulto Jovem
8.
Nat Chem Biol ; 13(5): 479-485, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28244988

RESUMO

Plant development requires coordination among complex signaling networks to enhance the plant's adaptation to changing environments. DELLAs, transcription regulators originally identified as repressors of phytohormone gibberellin signaling, play a central role in integrating multiple signaling activities via direct protein interactions with key transcription factors. Here, we found that DELLA is mono-O-fucosylated by the novel O-fucosyltransferase SPINDLY (SPY) in Arabidopsis thaliana. O-fucosylation activates DELLA by promoting its interaction with key regulators in brassinosteroid- and light-signaling pathways, including BRASSINAZOLE-RESISTANT1 (BZR1), PHYTOCHROME-INTERACTING-FACTOR3 (PIF3) and PIF4. Moreover, spy mutants displayed elevated responses to gibberellin and brassinosteroid, and increased expression of common target genes of DELLAs, BZR1 and PIFs. Our study revealed that SPY-dependent protein O-fucosylation plays a key role in regulating plant development. This finding may have broader importance because SPY orthologs are conserved in prokaryotes and eukaryotes, thus suggesting that intracellular O-fucosylation may regulate a wide range of biological processes in diverse organisms.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fucosiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fucosiltransferases/genética , Proteínas Repressoras/genética
9.
Nat Plants ; 3: 17010, 2017 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-28211915

RESUMO

The plant-specific GAI, RGA and SCR (GRAS) family proteins play critical roles in plant development and signalling. Two GRAS proteins, SHORT-ROOT (SHR) and SCARECROW (SCR), cooperatively direct asymmetric cell division and the patterning of root cell types by transcriptional control in conjunction with BIRD/INDETERMINATE DOMAIN (IDD) transcription factors, although precise details of these specific interactions and actions remain unknown. Here, we present the crystal structures of the SHR-SCR binary and JACKDAW (JKD)/IDD10-SHR-SCR ternary complexes. Each GRAS domain comprises one α/ß core subdomain with an α-helical cap that mediates heterodimerization by forming an intermolecular helix bundle. The α/ß core subdomain of SHR forms the BIRD binding groove, which specifically recognizes the zinc fingers of JKD. We identified a conserved SHR-binding motif in 13 BIRD/IDD transcription factors. Our results establish a structural basis for GRAS-GRAS and GRAS-BIRD interactions and provide valuable clues towards our understanding of these regulators, which are involved in plant-specific signalling networks.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Transporte/genética , Fatores de Transcrição/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
10.
Plant Physiol ; 173(2): 1463-1474, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28057895

RESUMO

PICKLE (PKL) is an ATP-dependent chromodomain-helicase-DNA-binding domain (CHD3) chromatin remodeling enzyme in Arabidopsis (Arabidopsis thaliana). Previous studies showed that PKL promotes embryonic-to-vegetative transition by inhibiting expression of seed-specific genes during seed germination. The pkl mutants display a low penetrance of the "pickle root" phenotype, with a thick and green primary root that retains embryonic characteristics. The penetrance of this pickle root phenotype in pkl is dramatically increased in gibberellin (GA)-deficient conditions. At adult stages, the pkl mutants are semidwarfs with delayed flowering time, which resemble reduced GA-signaling mutants. These findings suggest that PKL may play a positive role in regulating GA signaling. A recent biochemical analysis further showed that PKL and GA signaling repressors DELLAs antagonistically regulate hypocotyl cell elongation genes by direct protein-protein interaction. To elucidate further the role of PKL in GA signaling and plant development, we studied the genetic interaction between PKL and DELLAs using the hextuple mutant containing pkl and della pentuple (dP) mutations. Here, we show that PKL is required for most of GA-promoted developmental processes, including vegetative growth such as hypocotyl, leaf, and inflorescence stem elongation, and phase transitions such as juvenile-to-adult leaf and vegetative-to-reproductive phase. The removal of all DELLA functions (in the dP background) cannot rescue these phenotypes in pkl RNA-sequencing analysis using the ga1 (a GA-deficient mutant), pkl, and the ga1 pkl double mutant further shows that expression of 80% of GA-responsive genes in seedlings is PKL dependent, including genes that function in cell elongation, cell division, and phase transitions. These results indicate that the CHD3 chromatin remodeler PKL is required for regulating gene expression during most of GA-regulated developmental processes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , DNA Helicases/metabolismo , Giberelinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , DNA Helicases/genética , Regulação da Expressão Gênica de Plantas , Germinação , Família Multigênica , Mutação , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Sementes/genética , Sementes/crescimento & desenvolvimento , Transdução de Sinais
11.
Plant Physiol ; 171(4): 2760-70, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27255484

RESUMO

The phytohormone gibberellin (GA) plays a key role in promoting stem elongation in plants. Previous studies show that GA activates its signaling pathway by inducing rapid degradation of DELLA proteins, GA signaling repressors. Using an activation-tagging screen in a reduced-GA mutant ga1-6 background, we identified AtERF11 to be a novel positive regulator of both GA biosynthesis and GA signaling for internode elongation. Overexpression of AtERF11 partially rescued the dwarf phenotype of ga1-6 AtERF11 is a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of ERF/AP2 transcription factors in Arabidopsis (Arabidopsis thaliana). Overexpression of AtERF11 resulted in elevated bioactive GA levels by up-regulating expression of GA3ox1 and GA20ox genes. Hypocotyl elongation assays further showed that overexpression of AtERF11 conferred elevated GA response, whereas loss-of-function erf11 and erf11 erf4 mutants displayed reduced GA response. In addition, yeast two-hybrid, coimmunoprecipitation, and transient expression assays showed that AtERF11 enhances GA signaling by antagonizing the function of DELLA proteins via direct protein-protein interaction. Interestingly, AtERF11 overexpression also caused a reduction in the levels of another phytohormone ethylene in the growing stem, consistent with recent finding showing that AtERF11 represses transcription of ethylene biosynthesis ACS genes. The effect of AtERF11 on promoting GA biosynthesis gene expression is likely via its repressive function on ethylene biosynthesis. These results suggest that AtERF11 plays a dual role in promoting internode elongation by inhibiting ethylene biosynthesis and activating GA biosynthesis and signaling pathways.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Giberelinas/biossíntese , Caules de Planta/crescimento & desenvolvimento , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas , Modelos Biológicos , Caules de Planta/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética
12.
Genes Dev ; 30(2): 164-76, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26773002

RESUMO

The DELLA family of transcription regulators functions as master growth repressors in plants by inhibiting phytohormone gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also play a central role in mediating cross-talk between GA and other signaling pathways via antagonistic direct interactions with key transcription factors. However, how these crucial protein-protein interactions can be dynamically regulated during plant development remains unclear. Here, we show that DELLAs are modified by the O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) SECRET AGENT (SEC) in Arabidopsis. O-GlcNAcylation of the DELLA protein REPRESSOR OF ga1-3 (RGA) inhibits RGA binding to four of its interactors-PHYTOCHROME-INTERACTING FACTOR3 (PIF3), PIF4, JASMONATE-ZIM DOMAIN1, and BRASSINAZOLE-RESISTANT1 (BZR1)-that are key regulators in light, jasmonate, and brassinosteroid signaling pathways, respectively. Consistent with this, the sec-null mutant displayed reduced responses to GA and brassinosteroid and showed decreased expression of several common target genes of DELLAs, BZR1, and PIFs. Our results reveal a direct role of OGT in repressing DELLA activity and indicate that O-GlcNAcylation of DELLAs provides a fine-tuning mechanism in coordinating multiple signaling activities during plant development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas/genética , N-Acetilglucosaminiltransferases/metabolismo , Transdução de Sinais/fisiologia , Acilação , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Giberelinas/metabolismo , Mutação , N-Acetilglucosaminiltransferases/genética , Ligação Proteica
13.
Sensors (Basel) ; 15(11): 29408-18, 2015 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-26610504

RESUMO

Regular monitoring of blood α-fetoprotein (AFP) and/or carcino-embryonic antigen (CEA) levels is important for the routine screening of liver cancer. However, AFP and CEA have a much lower specificity than des-γ-carboxyprothrombin (DCP) to detect liver cancer. Therefore, the study reported here was designed, to develop a screen-printed DCP immunosensor incorporating zinc oxide nanoparticles, for accurate determination of DCP. The designed immunosensor shows low detection limits for the detection of DCP: 0.440 ng/mL (based on impedance measurement), 0.081 ng/mL (based on real part of impedance measurement) and 0.078 ng/mL (based on imaginary part of impedance measurement), within the range of 3.125 ng/mL to 2000 ng/mL. In addition, there was little interference to DCP determination by molecules such as Na⁺, K⁺, Ca(2+), Cl(-), glucose, urea, and uric acid. It is therefore concluded that the DCP immunosensor developed and reported here is simple, inexpensive and effective, and shows promise in the rapid screening of early-stage liver cancer at home with a point-of-care approach.


Assuntos
Biomarcadores Tumorais/sangue , Biomarcadores/sangue , Técnicas Biossensoriais/métodos , Técnicas Imunológicas/métodos , Neoplasias Hepáticas/diagnóstico , Nanopartículas Metálicas/química , Precursores de Proteínas/sangue , Animais , Bovinos , Desenho de Equipamento , Limite de Detecção , Modelos Lineares , Modelos Biológicos , Protrombina , Reprodutibilidade dos Testes , Soroalbumina Bovina , Óxido de Zinco/química
14.
J Exp Bot ; 66(5): 1463-76, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25588745

RESUMO

Gibberellins (GAs) regulate numerous developmental processes in grapevine (Vitis vinifera) such as rachis elongation, fruit set, and fruitlet abscission. The ability of GA to promote berry enlargement has led to its indispensable use in the sternospermocarpic ('seedless') table grape industry worldwide. However, apart from VvGAI1 (VvDELLA1), which regulates internode elongation and fruitfulness, but not berry size of seeded cultivars, little was known about GA signalling in grapevine. We have identified and characterized two additional DELLAs (VvDELLA2 and VvDELLA3), two GA receptors (VvGID1a and VvGID1b), and two GA-specific F-box proteins (VvSLY1a and VvSLY1b), in cv. Thompson seedless. With the exception of VvDELLA3-VvGID1b, all VvDELLAs interacted with the VvGID1s in a GA-dependent manner in yeast two-hybrid assays. Additionally, expression of these grape genes in corresponding Arabidopsis mutants confirmed their functions in planta. Spatiotemporal analysis of VvDELLAs showed that both VvDELLA1 and VvDELLA2 are abundant in most tissues, except in developing fruit where VvDELLA2 is uniquely expressed at high levels, suggesting a key role in fruit development. Our results further suggest that differential organ responses to exogenous GA depend on the levels of VvDELLA proteins and endogenous bioactive GAs. Understanding this interaction will allow better manipulation of GA signalling in grapevine.


Assuntos
Giberelinas/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Proteínas de Plantas/genética , Vitis/crescimento & desenvolvimento , Vitis/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Transdução de Sinais , Vitis/metabolismo
16.
Int J Nanomedicine ; 9: 3069-76, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25061289

RESUMO

The aims of this study were to investigate the most effective combination of physical forces from laser, electroporation, and reverse iontophoresis for noninvasive transdermal extraction of uric acid, and to develop a highly sensitive uric acid biosensor (UAB) for quantifying the uric acid extracted. It is believed that the combination of these physical forces has additional benefits for extraction of molecules other than uric acid from human skin. A diffusion cell with porcine skin was used to investigate the most effective combination of these physical forces. UABs coated with ZnO2 nanoparticles and constructed in an array configuration were developed in this study. The results showed that a combination of laser (0.7 W), electroporation (100 V/cm(2)), and reverse iontophoresis (0.5 mA/cm(2)) was the most effective and significantly enhanced transdermal extraction of uric acid. A custom-designed UAB coated with ZnO2 nanoparticles and constructed in a 1×3 array configuration (UAB-1×3-ZnO2) demonstrated enough sensitivity (9.4 µA/mM) for quantifying uric acid extracted by the combined physical forces of laser, electroporation, and RI. A good linear relationship (R(2)=0.894) was demonstrated to exist between the concentration of uric acid (0.2-0.8 mM) inside the diffusion cell and the current response of the UAB-1×3-ZnO2. In conclusion, a new approach to noninvasive transdermal extraction and quantification of uric acid has been established.


Assuntos
Análise Química do Sangue/métodos , Eletroporação/métodos , Iontoforese/métodos , Ácido Úrico/sangue , Animais , Análise Química do Sangue/instrumentação , Eletroporação/instrumentação , Iontoforese/instrumentação , Modelos Biológicos , Pele , Suínos
17.
Plant J ; 79(6): 1020-1032, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24961590

RESUMO

Gibberellins (GAs) play a critical role in fruit-set and fruit growth. Gibberellin is perceived by its nuclear receptors GA INSENSITIVE DWARF1s (GID1s), which then trigger degradation of downstream repressors DELLAs. To understand the role of the three GA receptor genes (GID1A, GID1B and GID1C) in Arabidopsis during fruit initiation, we have examined their temporal and spatial localization, in combination with analysis of mutant phenotypes. Distinct expression patterns are revealed for each GID1: GID1A is expressed throughout the whole pistil, while GID1B is expressed in ovules, and GID1C is expressed in valves. Functional study of gid1 mutant combinations confirms that GID1A plays a major role during fruit-set and growth, whereas GID1B and GID1C have specific roles in seed development and pod elongation, respectively. Therefore, in ovules, GA perception is mediated by GID1A and GID1B, while GID1A and GID1C are involved in GA perception in valves. To identify tissue-specific interactions between GID1s and DELLAs, we analyzed spatial expression patterns of four DELLA genes that have a role in fruit initiation (GAI, RGA, RGL1 and RGL2). Our data suggest that GID1A can interact with RGA and GAI in all tissues, whereas GID1C-RGL1 and GID1B-RGL2 interactions only occur in valves and ovules, respectively. These results uncover specific functions of each GID1-DELLA in the different GA-dependent processes that occur upon fruit-set. In addition, the distribution of GA receptors in valves along with lack of expression of GA biosynthesis genes in this tissue, strongly suggests transport of GAs from the developing seeds to promote fruit growth.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Giberelinas/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Flores/citologia , Flores/genética , Flores/crescimento & desenvolvimento , Frutas/citologia , Frutas/genética , Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Germinação , Modelos Biológicos , Mutação , Especificidade de Órgãos , Óvulo Vegetal/citologia , Óvulo Vegetal/genética , Óvulo Vegetal/crescimento & desenvolvimento , Fenótipo , Plantas Geneticamente Modificadas , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão , Sementes/citologia , Sementes/genética , Sementes/crescimento & desenvolvimento
18.
Biomed Mater Eng ; 24(1): 21-8, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24211878

RESUMO

In this study, a urea biosensor was prepared by the immobilization of urease onto the sensitive membrane of an extended-base bipolar junction transistor. The pH variation was used to detect the concentration of urea. The SnO2/ITO glass, fabricated by sputtering SnO2 on the conductive ITO glass, was used as a pH-sensitive membrane, which was connected with a commercial bipolar junction transistor device. The gels, fabricated by the poly vinyl alcohol with pendent styrylpyridinium groups, were used to immobilize the urease. This readout circuit, fabricated in a 0.35-um CMOS 2P4M process, operated at 3.3V supply voltage. This circuit occupied an area of 1.0 mm × 0.9 mm. The dynamic range of the urea biosensor was from 1.4 to 64 mg/dl at the 10 mM phosphate buffer solution and the sensitivity of this range was about 65.8 mV/pUrea. The effect of urea biosensors with different pH values was considered, and the characteristics of urea biosensors based on EBBJT were described.


Assuntos
Técnicas Biossensoriais , Eletroquímica , Ureia/química , Engenharia Biomédica , Eletrônica , Enzimas Imobilizadas/química , Desenho de Equipamento , Concentração de Íons de Hidrogênio , Teste de Materiais , Álcool de Polivinil , Compostos de Piridínio/química , Compostos de Estanho/química , Urease/química
19.
Sensors (Basel) ; 13(10): 14161-74, 2013 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-24152934

RESUMO

The purpose of this study was to develop novel nanoscale biosensors using titania nanotubes (TNTs) made by anodization. Titania nanotubes were produced on pure titanium sheets by anodization at room temperature. In this research, the electrolyte composition ethylene glycol 250 mL/NH4F 1.5 g/DI water 20 mL was found to produce the best titania nanotubes array films for application in amperometric biosensors. The amperometric results exhibit an excellent linearity for uric acid (UA) concentrations in the range between 2 and 14 mg/dL, with 23.3 (µA·cm-2)·(mg/dL)-1 UA sensitivity, and a correlation coefficient of 0.993. The glucose biosensor presented a good linear relationship in the lower glucose concentration range between 50 and 125 mg/dL, and the corresponding sensitivity was approximately 249.6 (µA·cm-2)·(100 mg/dL)-1 glucose, with a correlation coefficient of 0.973.


Assuntos
Técnicas Biossensoriais/instrumentação , Condutometria/instrumentação , Eletrodos , Glucose/análise , Nanopartículas Metálicas/química , Titânio/química , Ácido Úrico/análise , Desenho de Equipamento , Análise de Falha de Equipamento , Nanopartículas Metálicas/ultraestrutura , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Transdutores
20.
PLoS One ; 8(4): e61639, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23630604

RESUMO

OBJECTIVES: This study aims to investigate the electrical properties of lumbar paraspinal muscles (LPM) of patients with acute lower back pain (LBP) and to study a new approach, namely Electrical Impedance Myography (EIM), for reliable, low-cost, non-invasive, and real-time assessment of muscle-strained acute LBP. DESIGN: Patients with muscle-strained acute LBP (n = 30) are compared to a healthy reference group (n = 30). Electrical properties of LPM are studied. BACKGROUND: EIM is a novel technique under development for the assessment of neuromuscular disease. Therefore, it is speculated that EIM can be employed for the assessment of muscle-strained acute LBP. METHODS: Surface electrodes, in 2-electrode configurations, was used to measure the electrical properties of patient's and healthy subject's LPM at six different frequencies (0.02, 25.02, 50.02, 1000.02, 3000.02, and 5000.02 kHz), with the amplitude of the applied voltage limited to 200 mV. Parameters of impedance (Z), extracellular resistance (Re), intracellular resistance (Ri), and the ratio of extracellular resistance to intracellular resistance (Re/Ri) of LBP patient's and healthy subject's LPM were assessed to see if significant difference in values obtained in muscle-strained acute LBP patients existed. RESULTS: Intraclass correlation coefficient (ICC) showed that all measurements (ICC>0.96 for all studying parameters: Z, Re, Ri, and Re/Ri) had good reliability and validity. Significant differences were found on Z between LBP patient's and healthy subject's LPM at all studying frequencies, with p<0.05 for all frequencies. It was also found that Re (p<0.05) and Re/Ri (p<0.05) of LBP patient's LPM was significant smaller than that of healthy subjects while Ri (p<0.05) of LBP patient's LPM was significant greater than that of healthy subjects. No statistical significant difference was found between the left and right LPM of LBP patients and healthy subjects on the four studying parameters. CONCLUSION: EIM is a promising technique for assessing muscle-strained acute LBP.


Assuntos
Dor Lombar/fisiopatologia , Músculo Esquelético/fisiopatologia , Entorses e Distensões/fisiopatologia , Adulto , Temperatura Corporal , Estudos de Casos e Controles , Impedância Elétrica , Feminino , Humanos , Dor Lombar/patologia , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/lesões , Músculo Esquelético/patologia , Miografia , Reprodutibilidade dos Testes , Pele/fisiopatologia , Entorses e Distensões/patologia
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