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1.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360584

RESUMO

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 µM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Ácidos Hidroxâmicos/farmacologia , Alface/metabolismo , Proteínas de Plantas/metabolismo , Protoplastos/metabolismo , Tabaco/metabolismo , Divisão Celular , Genoma de Planta , Alface/efeitos dos fármacos , Alface/genética , Alface/crescimento & desenvolvimento , Células Vegetais , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Inibidores da Síntese de Proteínas/farmacologia , Protoplastos/efeitos dos fármacos , Tabaco/efeitos dos fármacos , Tabaco/genética , Tabaco/crescimento & desenvolvimento
2.
Artigo em Inglês | MEDLINE | ID: mdl-33620309

RESUMO

A Gram-stain-positive, facultatively anaerobic, rod-shaped, endospore-forming, oxidase-positive, and catalase-negative strain designated as BRMEA1T was isolated from the surface-sterilized Selaginella involvens roots. Growth of strain BRMEA1T was found to occur at pH 6.0-8.0 (optimum, pH 7.0), 15-50 °C (optimum, 25-30 °C) and in the absence of NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BRMEA1T formed a lineage within the genus Neobacillus (family Bacillaceae) and showed the highest sequence similarity to Neobacillus drentensis DSM 15600T (98.3 %) and Neobacillus fumarioli KCTC 13885T (98.2 %), and less than 98.2 % 16S rRNA gene sequence similarity to the other members of the genus Neobacillus. Whole-genome analysis of strain BRMEA1T comprised a circular chromosome (5 632 809 bp in size) with 38.5 mol% G+C content. Digital DNA-DNA hybridization analyses revealed that strain BRMEA1T showed 20.5 and 22.0% genomic DNA relatedness with the closest species, N. drentensis DSM 15600T and N. fumarioli KCTC 13885T, respectively. The whole-genome sequence of strain BRMEA1T showed the presence of 11 specific conserved signature indels for the genus Neobacillus. The major cellular fatty acids (>10 %) of strain BRMEA1T were found to be iso-C15 : 0 and anteiso-C15 : 0, while the major polar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Polyphasic analysis results revealed that BRMEA1T represents a novel species of the genus Neobacillus, with the proposed name Neobacillus endophyticus sp. nov. The type strain is BRMEA1T (=KCTC 43208T=CCTCC AB 2020071T).

3.
Plants (Basel) ; 9(12)2020 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-33297321

RESUMO

We aimed to develop a novel technology capable of rapidly selecting mutant plant cell lines. Salt resistance was chosen as a rapid selection trait that is easily applicable to protoplast-derived cell colonies. Mesophyll protoplasts were cultured in a medium supplemented with 0, 50, 100, 150, 200, 250, and 300 mM NaCl. At NaCl concentrations ≥ 100 mM, cell colony formation was strongly inhibited after 4 weeks of culture. Tobacco protoplasts irradiated with 0, 50, 100, 200, and 400 Gy were then cultured to investigate the effects of radiation intensity on cell division. The optimal radiation intensity was 50 Gy. To develop salt-resistant tobacco mutant plants, protoplasts irradiated with 50 Gy were cultured in a medium containing 100 mM NaCl. The efficiency of cell colony formation from these protoplasts was approximately 0.002%. A salt-resistant mutant callus was selected and proliferated in the same medium and then transferred to a shoot inducing medium for adventitious shoot formation. The obtained shoots were then cultured in a medium supplemented with 200 mM NaCl and developed into normal plantlets. This rapid selection technology for generating salt-resistant tobacco mutants will be useful for the development of crop varieties resistant to environmental stresses.

4.
Int J Mol Sci ; 21(22)2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-33182800

RESUMO

Histone acetylation plays an important role in plant growth and development. Here, we investigated the effect of sodium butyrate (NaB), a histone deacetylase inhibitor, on adventitious shoot formation from protoplast-derived calli and cotyledon explants of tobacco (Nicotiana benthamiana) and tomato (Solanum lycopersicum). The frequency of adventitious shoot formation from protoplast-derived calli was higher in shoot induction medium (SIM) containing NaB than in the control. However, the frequency of adventitious shoot formation from cotyledon explants of tobacco under the 0.1 mM NaB treatment was similar to that in the control, but it decreased with increasing NaB concentration. Unlike in tobacco, NaB decreased adventitious shoot formation in tomato explants in a concentration-dependent manner, but it did not have any effect on adventitious shoot formation in calli. NaB inhibited or delayed the expression of D-type cyclin (CYCD3-1) and shoot-regeneration regulatory gene WUSCHEL (WUS) in cotyledon explants of tobacco and tomato. However, compared to that in control SIM, the expression of WUS was promoted more rapidly in tobacco calli cultured in NaB-containing SIM, but the expression of CYCD3-1 was inhibited. In conclusion, the effect of NaB on adventitious shoot formation and expression of CYCD3-1 and WUS genes depended on the plant species and whether the effects were tested on explants or protoplast-derived calli.


Assuntos
Ácido Butírico/farmacologia , Lycopersicon esculentum/efeitos dos fármacos , Lycopersicon esculentum/crescimento & desenvolvimento , Tabaco/efeitos dos fármacos , Tabaco/crescimento & desenvolvimento , Cotilédone/efeitos dos fármacos , Cotilédone/genética , Cotilédone/crescimento & desenvolvimento , Ciclina D/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Lycopersicon esculentum/genética , Proteínas de Plantas/genética , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Especificidade da Espécie , Tabaco/genética
5.
Int J Mol Sci ; 21(15)2020 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-32722633

RESUMO

Enhancing the competence for plant regeneration in tissue culture studies is an important issue not only for efficient genetic transformation of commercial crops but also for the reproducibility of scientific reports. In this study, we investigated optimization of several tissue culture conditions including plant growth regulators, types and ages of explants, culture densities, and plant position in order to improve the competence of adventitious shoot formation of the tomato (Solanum lycopersicum cv. Micro-Tom). In addition, we examined the differential expression of D-type cyclin (CYCD3-1) and several shoot regeneration regulatory genes from hypocotyl and cotyledon explants of tomato during shoot organogenesis. A treatment of 1 mg L-1 Zeatin and 0.1 mg L-1 Indole-3-acetic acid (IAA) in Murashige and Skoog (MS) medium containing 3% sucrose was optimal for adventitious shoot formation from hypocotyl and cotyledon explants. The younger explants exhibited more shoot formation regardless of explant types. Additionally, those closest to the shoot apical meristem produced more shoots compared to the other regions in the hypocotyl and the cotyledon explants. Gene expression of CYCD3-1, SHOOT MERISTEMLESS (STM), and cytokinin dependent WUSCHEL (WUS) was significantly higher in younger explants than in older ones. Furthermore, an increase in CYCD3-1, STM, and WUS expression was evident at the distal part of hypocotyls and the proximal part of cotyledons compared to other regions. These differential gene expression profiles exhibited good agreement with the results of shoot formation obtained from diverse explants of tomato. These results suggest that temporal and spatial gene expression of shoot regeneration regulatory genes plays an important role in enhancing the competence and the reproducibility of adventitious shoot formation from tomato explants.


Assuntos
Cotilédone/metabolismo , Regulação da Expressão Gênica de Plantas , Hipocótilo/metabolismo , Lycopersicon esculentum/metabolismo , Proteínas de Plantas/biossíntese
6.
Sci Rep ; 9(1): 16354, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31704990

RESUMO

Biocontrol offers a promising alternative to synthetic fungicides for the control of a variety of pre- and post-harvest diseases of crops. Black rot, which is caused by the pathogenic fungus Ceratocytis fimbriata, is the most destructive post-harvest disease of sweet potato, but little is currently known about potential biocontrol agents for this fungus. Here, we isolated several microorganisms from the tuberous roots and shoots of field-grown sweet potato plants, and analyzed their ribosomal RNA gene sequences. The microorganisms belonging to the genus Pantoea made up a major portion of the microbes residing within the sweet potato plants, and fluorescence microscopy showed these microbes colonized the intercellular spaces of the vascular tissue in the sweet potato stems. Four P. dispersa strains strongly inhibited C. fimbriata mycelium growth and spore germination, and altered the morphology of the fungal hyphae. The detection of dead C. fimbriata cells using Evans blue staining suggested that these P. dispersa strains have fungicidal rather than fungistatic activity. Furthermore, P. dispersa strains significantly inhibited C. fimbriata growth on the leaves and tuberous roots of a susceptible sweet potato cultivar ("Yulmi"). These findings suggest that P. dispersa strains could inhibit black rot in sweet potato plants, highlighting their potential as biocontrol agents.


Assuntos
Ascomicetos/crescimento & desenvolvimento , Ipomoea batatas/imunologia , Pantoea/fisiologia , Controle Biológico de Vetores , Doenças das Plantas/prevenção & controle , Folhas de Planta/imunologia , Raízes de Plantas/imunologia , Resistência à Doença , Ipomoea batatas/microbiologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Raízes de Plantas/microbiologia
7.
Mol Biol Rep ; 46(5): 5073-5077, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31313130

RESUMO

One of the most crucial steps for preventing viral pandemics is the early detection of the causative virus on site. Various molecular and immunological approaches have been developed for virus detection. In this study, we investigated the utility of the recently introduced convection polymerase chain reaction (cPCR) platform for the rapid and sensitive detection of various animal viruses in the field, including the foot-and-mouth disease virus (FMDV) and avian influenza viruses (AIVs). Primer sets were designed to simultaneously detect two highly conserved regions of the FMDV, including the 5' untranslated region (5'-UTR) and 3D gene, and to specifically amplify the NP and hemagglutinin (HA) genes of H5 and H9 subtypes of AIVs. The portable cPCR system was able to amplify from as low as 1 to 10 copies of viral cDNAs in the singleplex mode and 10 to 100 copies of viral cDNAs in the duplex mode within 21 min. Thus, our data suggest that the cPCR protocols developed in this study are highly sensitive and enable quick detection of animal viruses in biological samples.


Assuntos
Reação em Cadeia da Polimerase/métodos , Viroses/diagnóstico , Viroses/genética , Animais , Aves/genética , Primers do DNA/genética , Vírus da Febre Aftosa/genética , Vírus da Influenza A/genética , Influenza Aviária/diagnóstico , Reação em Cadeia da Polimerase/instrumentação , Sensibilidade e Especificidade
8.
Proc Natl Acad Sci U S A ; 115(7): E1675-E1683, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29378957

RESUMO

Protein trafficking is a fundamental mechanism of subcellular organization and contributes to organellar biogenesis. AtCAP2 is an Arabidopsis homolog of the Mesembryanthemum crystallinum calcium-dependent protein kinase 1 adaptor protein 2 (McCAP2), a member of the syntaxin superfamily. Here, we show that AtCAP2 plays an important role in the conversion to the lytic vacuole (LV) during early plant development. The AtCAP2 loss-of-function mutant atcap2-1 displayed delays in protein storage vacuole (PSV) protein degradation, PSV fusion, LV acidification, and biosynthesis of several vacuolar proteins during germination. At the mature stage, atcap2-1 plants accumulated vacuolar proteins in the prevacuolar compartment (PVC) instead of the LV. In wild-type plants, AtCAP2 localizes to the PVC as a peripheral membrane protein and in the PVC compartment recruits glyceraldehyde-3-phosphate dehydrogenase C2 (GAPC2) to the PVC. We propose that AtCAP2 contributes to LV biogenesis during early plant development by supporting the trafficking of specific proteins involved in the PSV-to-LV transition and LV acidification during early stages of plant development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Sementes/crescimento & desenvolvimento , Vacúolos/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Germinação , Proteínas Associadas aos Microtúbulos/genética , Biogênese de Organelas , Transporte Proteico , Sementes/genética , Sementes/metabolismo , Vacúolos/genética
9.
Mol Plant ; 11(4): 568-583, 2018 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-29317286

RESUMO

Endocytosis and subsequent trafficking pathways are crucial for regulating the activity of plasma membrane-localized proteins. Depending on cellular and physiological conditions, the internalized cargoes are sorted at (and transported from) the trans-Golgi network/early endosome (TGN/EE) to the vacuole for degradation or recycled back to the plasma membrane. How this occurs at the molecular level remains largely elusive. Here, we provide evidence that the ENTH domain-containing protein AtECA4 plays a crucial role in recycling cargoes from the TGN/EE to the plasma membrane in Arabidopsis thaliana. AtECA4:sGFP primarily localized to the TGN/EE and plasma membrane (at low levels). Upon NaCl or mannitol treatment, AtECA4:sGFP accumulated at the TGN/EE at an early time point but was released from the TGN/EE to the cytosol at later time points. The ateca4 mutant showed higher resistance to osmotic stress and more sensitive to exogenous abscisic acid (ABA) than the wild type, as well as increased expression of ABA-inducible genes RD29A and RD29B. Consistently, ABCG25, a plasma membrane-localized ABA exporter, accumulated at the prevacuolar compartment in ateca4, indicating a defect in recycling to the plasma membrane. However, the role of AtECA4 in cargo recycling is not specific to ABCG25, as it also functions in the recycling of BRI1. These results suggest that AtECA4 plays a crucial role in the recycling of endocytosed cargoes from the TGN/EE to the plasma membrane.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/metabolismo , Endossomos/metabolismo , Rede trans-Golgi/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , ATPases Transportadoras de Cálcio/genética , Secas , Regulação da Expressão Gênica de Plantas , Pressão Osmótica , Transporte Proteico , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Salinidade
10.
Plant Physiol ; 174(3): 1576-1594, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28487479

RESUMO

Prenylated Rab acceptor1 (PRA1) functions in the recruitment of prenylated Rab proteins to their cognate organelles. Arabidopsis (Arabidopsis thaliana) contains a large number of proteins belonging to the AtPRA1 family. However, their physiological roles remain largely unknown. Here, we investigated the physiological role of AtPRA1.F4, a member of the AtPRA1 family. A T-DNA insertion knockdown mutant of AtPRA1.F4, atpra1.f4, was smaller in stature than parent plants and possessed shorter roots, whereas transgenic plants overexpressing HA:AtPRA1.F4 showed enhanced development of secondary roots and root hairs. However, both overexpression and knockdown plants exhibited increased sensitivity to high-salt stress, lower vacuolar Na+/K+-ATPase and plasma membrane ATPase activities, lower and higher pH in the vacuole and apoplast, respectively, and highly vesiculated Golgi apparatus. HA:AtPRA1.F4 localized to the Golgi apparatus and assembled into high-molecular-weight complexes. atpra1.f4 plants displayed a defect in vacuolar trafficking, which was complemented by low but not high levels of HA:AtPRA1.F4 Overexpression of HA:AtPRA1.F4 also inhibited protein trafficking at the Golgi apparatus, albeit differentially depending on the final destination or type of protein: trafficking of vacuolar proteins, plasma membrane proteins, and trans-Golgi network (TGN)-localized SYP61 was strongly inhibited; trafficking of TGN-localized SYP51 was slightly inhibited; and trafficking of secretory proteins and TGN-localized SYP41 was negligibly or not significantly inhibited. Based on these results, we propose that Golgi-localized AtPRA1.F4 is involved in the exit of many but not all types of post-Golgi proteins from the Golgi apparatus. Additionally, an appropriate level of AtPRA1.F4 is crucial for its function at the Golgi apparatus.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Complexo de Golgi/metabolismo , Prenilação de Proteína , Proteínas de Transporte Vesicular/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Membrana Celular/enzimologia , Membrana Celular/ultraestrutura , Técnicas de Silenciamento de Genes , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Proteínas de Membrana/metabolismo , Mutação/genética , Desenvolvimento Vegetal/efeitos dos fármacos , Desenvolvimento Vegetal/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Prenilação de Proteína/efeitos dos fármacos , Proteínas SNARE/metabolismo , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento , Cloreto de Sódio/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo
11.
Mol Plant ; 9(7): 1004-17, 2016 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-27142778

RESUMO

Aquaporin (AQP) is a water channel protein found in various subcellular membranes of both prokaryotic and eukaryotic cells. The physiological functions of AQPs have been elucidated in many organisms. However, understanding their biogenesis remains elusive, particularly regarding how they assemble into tetramers. Here, we investigated the amino acid residues involved in the tetramer formation of the Arabidopsis plasma membrane AQP AtPIP2;1 using extensive amino acid substitution mutagenesis. The mutant proteins V41A/E44A, F51A/L52A, F87A/I91A, F92A/I93A, V95A/Y96A, and H216A/L217A, harboring alanine substitutions in the transmembrane (TM) helices of AtPIP2;1 polymerized into multiple oligomeric complexes with a variable number of subunits greater than four. Moreover, these mutant proteins failed to traffic to the plasma membrane, instead of accumulating in the endoplasmic reticulum (ER). Structure-based modeling revealed that these residues are largely involved in interactions between TM helices within monomers. These results suggest that inter-TM interactions occurring both within and between monomers play crucial roles in tetramer formation in the AtPIP2;1 complex. Moreover, the assembly of AtPIP2;1 tetramers is critical for their trafficking from the ER to the plasma membrane, as well as water permeability.


Assuntos
Aquaporinas/química , Aquaporinas/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Aquaporinas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/genética , Retículo Endoplasmático/metabolismo , Multimerização Proteica/genética , Multimerização Proteica/fisiologia , Estrutura Secundária de Proteína
12.
Methods Mol Biol ; 1043: 113-20, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23913041

RESUMO

In eukaryotic cells, a large number of proteins are transported to their final destination after translation by a process called intracellular trafficking. Transient gene expression, either in plant protoplasts or in specific plant tissues, is a fast, flexible, and reproducible approach to study the cellular function of proteins, protein subcellular localizations, and protein-protein interactions. Here we describe the general method of protoplast isolation, polyethylene glycol-mediated protoplast transformation and immunostaining of protoplast or intact root tissues for studying the localization of protein in Arabidopsis.


Assuntos
Biologia Molecular/métodos , Raízes de Plantas/ultraestrutura , Protoplastos/ultraestrutura , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/ultraestrutura , Raízes de Plantas/química , Protoplastos/metabolismo
13.
Plant Physiol ; 161(1): 121-33, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23175753

RESUMO

In eukaryotic cells, protein trafficking plays an essential role in biogenesis of proteins that belong to the endomembrane compartments. In this process, an important step is the sorting of organellar proteins depending on their final destinations. For vacuolar proteins, vacuolar sorting receptors (VSRs) and receptor homology-transmembrane-RING H2 domain proteins (RMRs) are thought to be responsible. Arabidopsis (Arabidopsis thaliana) contains seven VSRs. Among them, VSR1, VSR3, and VSR4 are involved in sorting storage proteins targeted to the protein storage vacuole (PSV) in seeds. However, the identity of VSRs for soluble proteins of the lytic vacuole in vegetative cells remains controversial. Here, we provide evidence that VSR1, VSR3, and VSR4 are involved in sorting soluble lytic vacuolar and PSV proteins in vegetative cells. In protoplasts from leaf tissues of vsr1vsr3 and vsr1vsr4 but not vsr5vsr6, and rmr1rmr2 and rmr3rmr4 double mutants, soluble lytic vacuolar (Arabidopsis aleurain-like protein:green fluorescent protein [GFP] and carboxypeptidase Y:GFP and PSV (phaseolin) proteins, but not the vacuolar membrane protein Arabidopsis ßFructosidase4:GFP, exhibited defects in their trafficking; they accumulated to the endoplasmic reticulum with an increased secretion into medium. The trafficking defects in vsr1vsr4 protoplasts were rescued by VSR1 or VSR4 but not VSR5 or AtRMR1. Furthermore, of the luminal domain swapping mutants between VSR1 and VSR5, the mutant with the luminal domain of VSR1, but not that of VSR5, rescued the trafficking defects of Arabidopsis aleurain-like protein:GFP and phaseolin in vsr1vsr4 protoplasts. Based on these results, we propose that VSR1, VSR3, and VSR4, but not other VSRs, are involved in sorting soluble lytic vacuolar and PSV proteins for their trafficking to the vacuoles in vegetative cells.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Células Vegetais/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Vacúolos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Western Blotting , Retículo Endoplasmático/metabolismo , Teste de Complementação Genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Folhas de Planta/genética , Folhas de Planta/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Protoplastos/citologia , Protoplastos/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Solubilidade , Transformação Genética
14.
Plant Physiol ; 157(2): 645-58, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21828250

RESUMO

Prenylated Rab acceptors (PRAs), members of the Ypt-interacting protein family of small membrane proteins, are thought to aid the targeting of prenylated Rabs to their respective endomembrane compartments. In plants, the Arabidopsis (Arabidopsis thaliana) PRA1 family contains 19 members that display varying degrees of sequence homology to animal PRA1 and localize to the endoplasmic reticulum (ER) and/or endosomes. However, the exact role of these proteins remains to be fully characterized. In this study, the effect of AtPRA1.B6, a member of the AtPRA1 family, on the anterograde trafficking of proteins targeted to various endomembrane compartments was investigated. High levels of AtPRA1.B6 resulted in differential inhibition of coat protein complex II vesicle-mediated anterograde trafficking. The trafficking of the vacuolar proteins sporamin:GFP (for green fluorescent protein) and AALP:GFP, the secretory protein invertase:GFP, and the plasma membrane proteins PMP:GFP and H+-ATPase:GFP was inhibited in a dose-dependent manner, while the trafficking of the Golgi-localized proteins ST:GFP and KAM1(ΔC):mRFP was not affected. Conversely, in RNA interference plants displaying lower levels of AtPRA1.B6 transcripts, the trafficking efficiency of sporamin:GFP and AALP:GFP to the vacuole was increased. Localization and N-glycan pattern analyses of cargo proteins revealed that AtPRA1.B6-mediated inhibition of anterograde trafficking occurs at the ER. In addition, AtPRA1.B6 levels were controlled by cellular processes, including 26S proteasome-mediated proteolysis. Based on these results, we propose that AtPRA1.B6 is a negative regulator of coat protein complex II vesicle-mediated anterograde trafficking for a subset of proteins at the ER.


Assuntos
Proteínas de Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Arabidopsis/genética , Sequência de Bases , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Mutação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Interferência de RNA , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/genética , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismo
15.
Traffic ; 12(2): 185-200, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21059161

RESUMO

Prenylated Rab acceptors (PRAs) bind to prenylated Rab proteins and possibly aid in targeting Rabs to their respective compartments. In Arabidopsis, 19 isoforms of PRA1 have been identified and, depending upon the isoforms, they localize to the endoplasmic reticulum (ER), Golgi apparatus and endosomes. Here, we investigated the localization and trafficking of AtPRA1.B6, an isoform of the Arabidopsis PRA1 family. In colocalization experiments with various organellar markers, AtPRA1.B6 tagged with hemagglutinin (HA) at the N-terminus localized to the Golgi apparatus in protoplasts and transgenic plants. The valine residue at the C-terminal end and an EEE motif in the C-terminal cytoplasmic domain were critical for anterograde trafficking from the ER to the Golgi apparatus. The N-terminal region contained a sequence motif for retention of AtPRA1.B6 at the Golgi apparatus. In addition, anterograde trafficking of AtPRA1.B6 from the ER to the Golgi apparatus was highly sensitive to the HA:AtPRA1.B6 level. The region that contains the sequence motif for Golgi retention also conferred the abundance-dependent trafficking inhibition. On the basis of these results, we propose that AtPRA1.B6 localizes to the Golgi apparatus and its ER-to-Golgi trafficking and localization to the Golgi apparatus are regulated by multiple sequence motifs in both the C- and N-terminal cytoplasmic domains.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Complexo de Golgi/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Prenilação de Proteína/genética , Estrutura Terciária de Proteína , Transporte Proteico , Protoplastos/metabolismo , Relação Estrutura-Atividade , Proteínas rab de Ligação ao GTP/genética
16.
Mol Cells ; 22(2): 210-9, 2006 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-17085974

RESUMO

Dynamin-related protein 2A (AtDRP2A, formally ADL6), a member of the dynamin family, is critical for protein trafficking from the TGN to the central vacuole. However, the mechanism controlling its activity is not well understood in plant cells. We isolated Arabidopsis sec13 homolog1 (AtSeh1) that interacts with AtDRP2A by a yeast two-hybrid screening. AtSeh1 has four WD40 motifs and amino acid sequence homology to Sec13, a component of COPII vesicles. Coimmunoprecipitation and protein pull-down experiments demonstrated specific interaction between AtSeh1 and AtDRP2A. AtSeh1 bound to the pleckstrin homology domain of AtDRP2A in competition with the C-terminal domain of the latter, and this resulted in inhibition of the interaction between AtDRP2A and PtdIns3P in vitro. AtSeh1 localized to multiple locations: the nucleus, the prevacuolar compartment and the Golgi complex. Based on these results we propose that AtSeh1 plays a role in regulating cycling of AtDRP2A between membrane-bound and soluble forms.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Dinaminas/metabolismo , Vacúolos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/química , Núcleo Celular/metabolismo , Dinaminas/química , Complexo de Golgi/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transporte Proteico , Alinhamento de Sequência , Técnicas do Sistema de Duplo-Híbrido
17.
Plant Cell ; 18(9): 2258-74, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16905657

RESUMO

Epsin and related proteins play important roles in various steps of protein trafficking in animal and yeast cells. Many epsin homologs have been identified in plant cells from analysis of genome sequences. However, their roles have not been elucidated. Here, we investigate the expression, localization, and biological role in protein trafficking of an epsin homolog, Arabidopsis thaliana EPSIN1, which is expressed in most tissues we examined. In the cell, one pool of EPSIN1 is associated with actin filaments, producing a network pattern, and a second pool localizes primarily to the Golgi complex with a minor portion to the prevacuolar compartment, producing a punctate staining pattern. Protein pull-down and coimmunoprecipitation experiments reveal that Arabidopsis EPSIN1 interacts with clathrin, VTI11, gamma-adaptin-related protein (gamma-ADR), and vacuolar sorting receptor1 (VSR1). In addition, EPSIN1 colocalizes with clathrin and VTI11. The epsin1 mutant, which has a T-DNA insertion in EPSIN1, displays a defect in the vacuolar trafficking of sporamin:green fluorescent protein (GFP), but not in the secretion of invertase:GFP into the medium. Stably expressed HA:EPSIN1 complements this trafficking defect. Based on these data, we propose that EPSIN1 plays an important role in the vacuolar trafficking of soluble proteins at the trans-Golgi network via its interaction with gamma-ADR, VTI11, VSR1, and clathrin.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Clatrina/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Complexo 1 de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Complexo de Golgi/metabolismo , Transporte Proteico/genética , Transporte Proteico/fisiologia , Proteínas Qb-SNARE/metabolismo , Solubilidade
18.
FEBS Lett ; 556(1-3): 127-36, 2004 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-14706839

RESUMO

Phosphoinositide-specific phospholipase C (PI-PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate and diacylglycerol, both of which act as secondary messengers in animal cells. In this report, we identified in Vigna radiata L. (mung bean) three distinct partial cDNAs (pVr-PLC1, pVr-PLC2, and pVr-PLC3), which encode forms of putative PI-PLC. All three Vr-PLC genes were transcriptionally active and displayed unique patterns of expression. The Vr-PLC1 and Vr-PLC2 transcripts were constitutively expressed to varying degrees in every tissue of mung bean plants examined. In contrast, the Vr-PLC3 mRNA level was very low under normal growth conditions and was rapidly induced in an abscisic acid-independent manner under environmental stress conditions (drought and high salinity). An isolated genomic clone, about 8.2 kb in length, showed that Vr-PLC1 and Vr-PLC3 are in tandem array in the mung bean genome. The predicted primary sequence of Vr-PLC3 (M(r)=67.4 kDa) is reminiscent of the delta-isoform of animal enzymes which contain core sequences found in typical PI-PLCs, such as the catalytic domain comprising X and Y motifs, a lipid-binding C2 domain, and the less conserved EF-hand domain. Results of in vivo targeting experiment using a green fluorescent protein (GFP) showed that the GFP-Vr-PLC3 fusion protein was localized primarily to the plasma membrane of the Arabidopsis protoplast. The C2 domain was essential for Vr-PLC3 to be targeted to the plasma membrane. The possible biological functions of stress-responsive Vr-PLC3 in mung bean plants are discussed.


Assuntos
Fabaceae/enzimologia , Fabaceae/genética , Genes de Plantas , Fosfatidilinositol Diacilglicerol-Liase/biossíntese , Fosfatidilinositol Diacilglicerol-Liase/genética , Sequência de Aminoácidos , Membrana Celular/enzimologia , Sequência Conservada , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Isoenzimas/biossíntese , Isoenzimas/genética , Cinética , Manitol/farmacologia , Dados de Sequência Molecular , Fosfoinositídeo Fosfolipase C , Filogenia , Estruturas Vegetais/genética , Estruturas Vegetais/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologia
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