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1.
Front Plant Sci ; 12: 702930, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34267775

RESUMO

Seed oils are used as edible oils and increasingly also for industrial applications. Although high-oleic seed oil is preferred for industrial use, most seed oil is high in polyunsaturated fatty acids (PUFAs) and low in monounsaturated fatty acids (MUFAs) such as oleic acid. Oil from Camelina, an emerging oilseed crop with a high seed oil content and resistance to environmental stress, contains 60% PUFAs and 30% MUFAs. Hexaploid Camelina carries three homoeologs of FAD2, encoding fatty acid desaturase 2 (FAD2), which is responsible for the synthesis of linoleic acid from oleic acid. In this study, to increase the MUFA contents of Camelina seed oil, we generated CsFAD2 knockout plants via CRISPR-Cas9-mediated gene editing using the pRedU6fad2EcCas9 vector containing DsRed as a selection marker, the U6 promoter to drive a single guide RNA (sgRNA) covering the common region of the three CsFAD2 homoeologs, and an egg-cell-specific promoter to drive Cas9 expression. We analyzed CsFAD2 homoeolog-specific sequences by PCR using genomic DNA from transformed Camelina leaves. Knockout of all three pairs of FAD2 homoeologs led to a stunted bushy phenotype, but greatly enhanced MUFA levels (by 80%) in seeds. However, transformants with two pairs of CsFAD2 homoeologs knocked out but the other pair wild-type heterozygous showed normal growth and a seed MUFAs production increased up to 60%. These results provide a basis for the metabolic engineering of genes that affect growth in polyploid crops through genome editing.

2.
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.

3.
Front Plant Sci ; 11: 610251, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33510753

RESUMO

Rare cold inducible 2 (RCI2) proteins are small hydrophobic membrane proteins in plants, and it has been widely reported that RCI2 expressions are dramatically induced by salt, cold, and drought stresses in many species. The RCI2 proteins have been shown to regulate plasma membrane (PM) potential and enhance abiotic stress tolerance when over-expressed in plants. RCI2 protein structures contain two transmembrane domains that are thought to be PM intrinsic proteins and have been observed at the PM and endomembranes. However, cellular trafficking of RCI2s are not fully understood. In this review, we discussed (i) general properties of RCI2s characterized in many species, (ii) the uses of RCI2s as a tracer in live cell imaging analyses and when they are fused to fluorescence proteins during investigations into vesicle trafficking, and (iii) RCI2 functionalities such as their involvement in rapid diffusion, endocytosis, and protein interactions. Consequently, the connection between physiological characteristics of RCI2s and traffic of RCI2s interacting membrane proteins might be helpful to understand role of RCI2s contributing abiotic stresses tolerance.

4.
J Plant Physiol ; 240: 153011, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31357099

RESUMO

Phytochelatin synthase (PCS) is an enzyme that synthesizes phytochelatins, which are metal-binding peptides. Despite the important role of PCS in heavy metal detoxification or tolerance, the functional role of PCS with respect to other abiotic stresses remains largely unknown. In this study, we determined the function of Arabidopsis thaliana phytochelatin synthase 2 (AtPCS2) in the salt stress response. Expression of AtPCS2 was significantly increased in response to 100 and 200 mM NaCl treatment. AtPCS2-overexpressing transgenic Arabidopsis and tobacco plants displayed increased seed germination rates and seedling growth under high salt stress. In addition, transgenic Arabidopsis subjected to salt stress exhibited enhanced proline accumulation and reduced Na+/K+ ratios compared to wild type plants. Furthermore, decreased levels of hydrogen peroxide (H2O2) and lipid peroxidation were observed in transgenic Arabidopsis compared to wild type specimens. Salt stress greatly reduced transcript levels of CuSOD2, FeSOD2, CAT2, and GR2 in wild type but not transgenic Arabidopsis. Notably, levels of CAT3 in transgenic Arabidopsis were markedly increased upon salt stress, suggesting that low accumulation of H2O2 in transgenic Arabidopsis is partially achieved through induction of CAT. Collectively, these results suggest that AtPCS2 plays a positive role in seed germination and seedling growth under salt stress through a series of indirect effects that are likely involved in H2O2 scavenging, regulation of osmotic adjustment and ion homeostasis.


Assuntos
Aminoaciltransferases/genética , Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/fisiologia , Tolerância ao Sal/genética , Cloreto de Sódio/farmacologia , Tabaco/fisiologia , Aminoaciltransferases/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relação Dose-Resposta a Droga , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Tabaco/efeitos dos fármacos , Tabaco/enzimologia , Tabaco/genética
5.
Biochem Biophys Res Commun ; 513(1): 213-218, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30954220

RESUMO

Rare cold-inducible 2 (RCI2) proteins are small hydrophobic proteins that are known to be localized in cellular membranes. The function of RCI2 proteins has been reported to be associated with low-temperature, salt, and drought stress tolerances as a membrane potential regulator; however, the specific functions are still unknown. The PIP2 (plasma membrane intrinsic protein 2) aquaporins are proteins that transport water and small solutes into the cell. The expression and activity of PIP2 proteins, like RCI2, are also related to salt- and drought-stress tolerance. In this study, we identified novel protein interactions between RCI2 and PIP2; 1, including protein accumulation changes in the bioenergy crop Camelina sativa L. under various NaCl stress conditions. Accumulation of both CsRCI2E and CsRCI2F proteins increased with NaCl stress; however, to differing levels depending on the NaCl stress intensity. A co-immunoprecipitation test revealed interaction between CsRCI2E-CsPIP2 and CsRCI2F-CsPIP2. Moreover, co-expression of the four CsRCI2 proteins with CsPIP2; 1 in Xenopus laevis oocytes reduced water transport activity. Furthermore, the abundance of CsPIP2; 1 protein was decreased under CsRCI2E and CsRCI2F co-expression. These results suggest that NaCl-induced expression of CsRCI2E and CsRCI2F contributes to the regulation of CsPIP2; 1.


Assuntos
Aquaporinas/metabolismo , Brassicaceae/fisiologia , Proteínas de Plantas/metabolismo , Estresse Salino , Água/metabolismo , Animais , Secas , Mapas de Interação de Proteínas , Cloreto de Sódio/metabolismo , Xenopus
6.
Plant Cell Rep ; 34(9): 1489-98, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25972262

RESUMO

KEY MESSAGE: Under heavy-metal stress, CsHMA3 overexpressing transgenic Camelina plants displayed not only a better quality, but also a higher quantity of unsaturated fatty acids in their seeds compared with wild type. Camelina sativa L. belongs to the Brassicaceae family and is frequently used as a natural vegetable oil source, as its seeds contain a high content of fatty acids. In this study, we observed that, when subjected to heavy metals (Cd, Co, Zn and Pb), the seeds of CsHMA3 (Heavy-Metal P1B-ATPase 3) transgenic lines retained their original golden yellow color and smooth outline, unlike wild-type seeds. Furthermore, we investigated the fatty acids content and composition of wild type and CsHMA3 transgenic lines after heavy metal treatments compared to the control. The results showed higher total fatty acid amounts in seeds of CsHMA3 transgenic lines compared with those in wild-type seeds under heavy-metal stresses. In addition, the compositions of unsaturated fatty acids-especially 18:1 (oleic acid), 18:2 (linoleic acid; only in case of Co treatment), 18:3 (linolenic acid) and 20:1 (eicosenoic acid)-in CsHMA3 overexpressing transgenic lines treated with heavy metals were higher than those of wild-type seeds under the same conditions. Furthermore, reactive oxygen species (ROS) contents in wild-type leaves and roots when treated with heavy metal were higher than in CsHMA3 overexpressing transgenic lines. These results indicate that overexpression of CsHMA3 affects fatty acid composition and content-factors that are responsible for the fuel properties of biodiesel-and can alleviate ROS accumulation caused by heavy-metal stresses in Camelina. Due to these factors, we propose that CsHMA3 transgenic Camelina can be used for phytoremediation of metal-contaminated soil as well as for oil production.


Assuntos
Brassicaceae/metabolismo , Ácidos Graxos/metabolismo , Metais Pesados/toxicidade , Proteínas de Plantas/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Antocianinas/metabolismo , Brassicaceae/efeitos dos fármacos , Brassicaceae/genética , Clorofila/metabolismo , Germinação/efeitos dos fármacos , Fenótipo , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio/metabolismo , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento
7.
J Plant Physiol ; 171(15): 1401-12, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25046761

RESUMO

Aquaporin (AQP) proteins are involved in water homeostasis in cells at all taxonomic levels of life. Phosphorylation of some AQPs has been proposed to regulate water permeability via gating of the channel itself. We analyzed plasma membrane intrinsic proteins (PIP) from Camelina and characterized their biological functions under both stressful and favorable conditions. A three-dimensional theoretical model of the Camelina AQP proteins was built by homology modeling which could prove useful in further functional characterization of AQPs. CsPIP2;1 was strongly and constitutively expressed in roots and leaves of Camelina, suggesting that this gene is related to maintenance of homeostasis during salt and drought stresses. CsPIP2s exhibited water channel activity in Xenopus oocytes. We then examined the roles of CsPIP2;1 phosphorylation at Ser273 and Ser277 in the regulation of water permeability using phosphorylation mutants. A single deletion strain of CsPIP2;1 was generated to serve as the primary host for testing AQP expression constructs. A Ser277 to alanine mutation (to prevent phosphorylation) did not change CsPIP2;1 water permeability while a Ser273 mutation to alanine did affect water permeability. Furthermore, a CsPIP2;1 point mutation when ectopically expressed in yeast resulted in lower growth in salt and drought conditions compared with controls, and confirmation of Ser273 as the phosphorylation site. Our results support the idea that post-translational modifications in the Ser273 regulatory domains of the C-terminus fine tune water flux through CsPIP2;1.


Assuntos
Aquaporinas/metabolismo , Brassicaceae/fisiologia , Regulação da Expressão Gênica de Plantas , Estresse Fisiológico , Sequência de Aminoácidos , Animais , Aquaporinas/genética , Brassicaceae/efeitos dos fármacos , Brassicaceae/genética , Secas , Feminino , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Oócitos , Fosforilação , Filogenia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Alinhamento de Sequência , Serina , Cloreto de Sódio/farmacologia , Água/metabolismo , Xenopus
8.
Biotechnol Biofuels ; 7: 96, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25018780

RESUMO

BACKGROUND: Camelina sativa (L.) Crantz, known by such popular names as "gold-of-pleasure" and "false flax," is an alternative oilseed crop for biofuel production and can be grown in harsh environments. Considerable interest is now being given to the new concept of the development of a fusion plant which can be used as a soil remediation plant for ground contaminated by heavy metals as well as a bioenergy crop. However, knowledge of the transport processes for heavy metals across Camelina plant membranes is still rudimentary. RESULTS: Firstly, to investigate whether Camelina HMA (heavy metal P1B-ATPase) genes could be used in such a plant, we analyzed the expression patterns of eight HMA genes in Camelina (taken from the root, leaf, stem, flower, and silique). CsHMA3 genes were expressed in all organs. In addition, CsHMA3 was induced in roots and leaves especially after Pb treatment. Heterogeneous expression of CsHMA3 complemented the Pb- or Zn-sensitive phenotype of Δycf1 or Δzrc1 yeast mutant strains. Subsequently, we cloned and overexpressed CsHMA3 in Camelina. The root growth of transgenic lines was better than that in the wild-type plant under heavy metal stress (for Cd, Pb, and Zn). In particular, the transgenic lines showed enhanced Pb tolerance in a wide range of Pb concentrations. Furthermore, the Pb and Zn content in the shoots of the transgenic lines were higher than those in the wild-type plant. These results suggest that overexpression of CsHMA3 might enhance Pb and Zn tolerance and translocation. Also, the transgenic lines displayed a wider leaf shape compared with the wild-type plant due to an induction of genes related to leaf width growth and showed a greater total seed yield compared to the wild type under heavy metal stress. CONCLUSIONS: Our data obtained from physiological and functional analyses using CsHMA3 overexpression plants will be useful to develop a multifunctional plant that can improve the productivity of a bioenergy crop and simultaneously be used to purify an area contaminated by various heavy metals.

9.
Plant Physiol Biochem ; 68: 44-51, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23628924

RESUMO

Camelina sativa L. is an oil-seed crop that has potential for biofuel applications. Although the importance of C. sativa as a biofuel crop has increased in recent years, reports demonstrating the stress responsiveness of C. sativa and characterizing the genes involved in stress response of C. sativa have never been published. Here, we isolated and characterized three genes encoding glycine-rich RNA-binding proteins (GRPs) from camelina: CsGRP2a, CsGRP2b, and CsGRP2c. The three CsGRP2 proteins were very similar in amino acid sequence and contained a well-conserved RNA-recognition motif at the N-terminal region and glycine-rich domain at the C-terminal region. To understand the functional roles of CsGRP2s under stress conditions, we investigated the expression patterns of CsGRP2s under various environmental stress conditions. The expressions of the three CsGRP2s were highly up-regulated under cold stress. The expression of CsGRP2a was up-regulated under salt or dehydration stress, whereas the transcript levels of CsGRP2b and CsGRP2c were decreased under salt or dehydration stress conditions. The three CsGRP2s had the ability to complement cold-sensitive Escherichia coli mutants at low temperatures and harbored transcription anti-termination and nucleic acid-melting activities, indicating that the CsGRP2s possess RNA chaperone activity. The CsGRP2a protein was localized to both the nucleus and the cytoplasm. Expression of CsGRP2a in cold-sensitive Arabidopsis grp7 mutant plants resulted in decreased electrolyte leakage at freezing temperatures. Collectively, these results suggest that the stress-responsive CsGRP2s play a role as an RNA chaperone during the stress adaptation process in camelina.


Assuntos
Brassicaceae/fisiologia , Proteínas de Plantas/genética , Proteínas de Ligação a RNA/genética , Estresse Fisiológico/genética , Adaptação Fisiológica/genética , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brassicaceae/genética , Clonagem Molecular , Resposta ao Choque Frio/genética , Escherichia coli/genética , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Homologia de Sequência de Aminoácidos , Regulação para Cima
10.
J Plant Physiol ; 170(11): 1028-38, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23537705

RESUMO

Jatropha has potential to be an important bio-fuel crop due to such advantages as high seed oil content and the ability to grow well on marginal lands less suited for food crops. Despite its ability to grow on marginal land, Jatropha is still susceptible to high salt and drought stresses, which can significantly reduce plant growth, stomatal conductance, sap-flow rate, and plant sap volume. This study was undertaken to collect basic knowledge of the physiological and molecular aspects of Jatropha response to salt and drought stresses, and to elucidate how Jatropha recovers from stress. From these studies we identified candidate genes that may be useful for the development of Jatropha cultivars that will grow efficiently in arid and barren lands. Of particular interest, two plasma membrane intrinsic proteins were identified: Jatropha plasma membrane intrinsic protein 1 (JcPIP1) and Jatropha plasma membrane intrinsic protein 2 (JcPIP2). The expression levels of JcPIP1 were dramatically increased in roots, stems, and leaves during the recovery from stress, whereas the JcPIP2 gene transcripts levels were induced in roots and stems during the water deficit stress. The protein levels of JcPIP1 and JcPIP2 were consistent with the gene expression patterns. Based on these results, we hypothesized that JcPIP1 plays a role in the recovery events from water stresses, while JcPIP2 is important in early responses to water stress. Virus induced gene silencing technology revealed that both JcPIP1 and JcPIP2 have positive roles in response to water deficit stresses, but have antagonistic functions at the recovery stage. We suggest that both JcPIP1 and JcPIP2 may play important roles in responses to water deficit conditions and both have potential as targets for genetic engineering.


Assuntos
Jatropha/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Plantas/metabolismo , Secas , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Jatropha/genética , Jatropha/fisiologia , Proteínas de Membrana/genética , Proteínas de Plantas/genética
11.
J Plant Physiol ; 170(9): 828-37, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23399403

RESUMO

Camelina (Camelina sativa) and rapeseed (Brassica napus) are well-established oil-seed crops with great promise also for biofuels. Both are cold-tolerant, and camelina is regarded to be especially appropriate for production on marginal lands. We examined physiological and biochemical alterations in both species during cold stress treatment for 3 days and subsequent recovery at the temperature of 25°C for 0, 0.25, 0.5, 1, 2, 6, and 24h, with particular emphasis on the post-translational regulation of the plasma membrane (PM) H(+)-ATPase (EC3.6.3.14). The activity and translation of the PM H(+)-ATPase, as well as 14-3-3 proteins, increased after 3 days of cold stress in both species but recovery under normal conditions proceeded differently. The increase in H(+)-ATPase activity was the most dramatic in camelina roots after recovery for 2h at 25°C, followed by decay to background levels within 24h. In rapeseed, the change in H(+)-ATPase activity during the recovery period was less pronounced. Furthermore, H(+)-pumping increased in both species after 15min recovery, but to twice the level in camelina roots compared to rapeseed. Protein gel blot analysis with phospho-threonine anti-bodies showed that an increase in phosphorylation levels paralleled the increase in H(+)-transport rate. Thus our results suggest that cold stress and recovery in camelina and rapeseed are associated with PM H(+)-fluxes that may be regulated by specific translational and post-translational modifications.


Assuntos
Brassica rapa/enzimologia , Brassicaceae/enzimologia , Regulação da Expressão Gênica de Plantas , ATPases Translocadoras de Prótons/metabolismo , Estresse Fisiológico/fisiologia , Transporte Biológico , Brassica rapa/fisiologia , Brassicaceae/fisiologia , Membrana Celular/enzimologia , Clorofila/metabolismo , Temperatura Baixa , Fosforilação , Folhas de Planta/enzimologia , Folhas de Planta/fisiologia , Raízes de Plantas/enzimologia , Raízes de Plantas/fisiologia , Estômatos de Plantas/enzimologia , Estômatos de Plantas/fisiologia , Transpiração Vegetal/fisiologia , Plântula/enzimologia , Plântula/fisiologia
12.
Bioresour Technol ; 130: 211-7, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23306131

RESUMO

The present study investigated the feasibility of the recovery and reuse oxalic acid in a multistage process for the pretreatment of a lignocellulosic biomass. Electrodialysis (ED), an electrochemical process using ion exchange membranes, was used to recover and reuse oxalic acid in the multistage process. The ED optimal condition for recover oxalic acid was potential of 10V and pH 2.2 in synthetic solutions. The recovery efficiency of oxalic acid from hydrolysates reached 100% at potential of 10V. The power consumption to treat 1mol of oxalic acid was estimated to be 41.0wh. At the same time, ethanol production increased up to 19g/L in the ED-treated hydrolysate, corresponding to ethanol productivity of 0.27g/L/h. It was clearly shown that bioethanol fermentation efficiency increased using the ED process, due to a small loss of fermentable sugar and a significantly high removal of inhibitory chemicals.


Assuntos
Biocombustíveis , Lignina/química , Ácido Oxálico/isolamento & purificação , Agaricales/crescimento & desenvolvimento , Biomassa , Diálise , Etanol/análise , Estudos de Viabilidade , Fermentação , Concentração de Íons de Hidrogênio , Ácido Oxálico/química
13.
PLoS One ; 7(8): e43707, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22912901

RESUMO

U12 intron-specific spliceosomes contain U11 and U12 small nuclear ribonucleoproteins and mediate the removal of U12 introns from precursor-mRNAs. Among the several proteins unique to the U12-type spliceosomes, an Arabidopsis thaliana AtU11/U12-31K protein has been shown to be indispensible for proper U12 intron splicing and for normal growth and development of Arabidopsis plants. Here, we assessed the functional roles of the rice (Oryza sativa) OsU11/U12-31K protein in U12 intron splicing and development of plants. The U11/U12-31K transcripts were abundantly expressed in the shoot apical meristems (SAMs) of Arabidopsis and rice. Ectopic expression of OsU11/U12-31K in AtU11/U12-31K-defecient Arabidopsis mutant complemented the incorrect U12 intron splicing and abnormal development phenotypes of the Arabidopsis mutant plants. Impaired cell division activity in the SAMs and inflorescence stems observed in the AtU11/U12-31K-deficient mutant was completely recovered to normal by the expression of OsU11/U12-31K. Similar to Arabidopsis AtU11/U12-31K, rice OsU11/U12-31K was determined to harbor RNA chaperone activity. Collectively, the present findings provide evidence for the emerging idea that the U11/U12-31K protein is an indispensible RNA chaperone that functions in U12 intron splicing and is necessary for normal development of monocotyledonous plants as well as dicotyledonous plants.


Assuntos
Íntrons/genética , Proteínas de Plantas/genética , Splicing de RNA , Ribonucleoproteínas Nucleares Pequenas/genética , Spliceossomos/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Divisão Celular/genética , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Hibridização In Situ , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Microscopia Eletrônica de Varredura , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Oryza/genética , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Interferência de RNA , RNA de Plantas/genética , RNA de Plantas/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo
15.
J Plant Res ; 125(6): 793-804, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22610130

RESUMO

Contrary to extensive researches on the roles of metallothioneins (MTs) in metal tolerance of animals, the roles of plant MTs in metal tolerance are largely under investigation. In this study, we evaluated the functional role of type 2 MT from Colocasia esculenta (CeMT2b) in Zn tolerance of tobacco and E. coli cells. Under Zn-stress conditions, transgenic tobacco overexpressing CeMT2b displayed much better seedling growth, a significant decrease in the levels of H(2)O(2) and an increase in Zn accumulation compared with the wild type. Overexpression of CeMT2b in E. coli greatly enhanced Zn tolerance and Zn accumulation under Zn stresses compared with control cells. CeMT2b bound 5.38 ± 0.29 atoms of Zn per protein. To identify a structural domain of CeMT2b for Zn binding, we investigated the growth of E. coli expressing each of the N-terminal, C-terminal, and central linker domains or a CNC motif deletion from the C-terminus of full-length CeMT2b. The results showed that the CNC motif is required for Zn tolerance, and the N-terminal domain is more effective in Zn tolerance than the C-terminal domain. Taken together, our results provide direct evidence for functional contributions of CeMT2b in Zn tolerance of tobacco and E. coli cells.


Assuntos
Colocasia/genética , Genes de Plantas , Metalotioneína/metabolismo , Tabaco/efeitos dos fármacos , Zinco/farmacologia , Motivos de Aminoácidos , Clonagem Molecular , Meios de Cultura/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Peróxido de Hidrogênio/metabolismo , Metalotioneína/genética , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Estrutura Terciária de Proteína , Espécies Reativas de Oxigênio/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Alinhamento de Sequência , Tabaco/genética , Tabaco/crescimento & desenvolvimento , Zinco/metabolismo
16.
Plant Physiol ; 159(1): 479-88, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22434042

RESUMO

The effects of low root temperature on growth and root cell water transport were compared between wild-type Arabidopsis (Arabidopsis thaliana) and plants overexpressing plasma membrane intrinsic protein 1;4 (PIP1;4) and PIP2;5. Descending root temperature from 25°C to 10°C quickly reduced cell hydraulic conductivity (L(p)) in wild-type plants but did not affect L(p) in plants overexpressing PIP1;4 and PIP2;5. Similarly, when the roots of wild-type plants were exposed to 10°C for 1 d, L(p) was lower compared with 25°C. However, there was no effect of low root temperature on L(p) in PIP1;4- and PIP2;5-overexpressing plants after 1 d of treatment. When the roots were exposed to 10°C for 5 d, L(p) was reduced in wild-type plants and in plants overexpressing PIP1;4, whereas there was still no effect in PIP2;5-overexpressing plants. These results suggest that the gating mechanism in PIP1;4 may be more sensitive to prolonged low temperature compared with PIP2;5. The reduction of L(p) at 10°C in roots of wild-type plants was partly restored to the preexposure level by 5 mm Ca(NO(3))(2) and protein phosphatase inhibitors (75 nm okadaic acid or 1 µm Na(3)VO(4)), suggesting that aquaporin phosphorylation/dephosphorylation processes were involved in this response. The temperature sensitivity of cell water transport in roots was reflected by a reduction in shoot and root growth rates in the wild-type and PIP1;4-overexpressing plants exposed to 10°C root temperature for 5 d. However, low root temperature had no effect on growth in plants overexpressing PIP2;5. These results provide strong evidence for a link between growth at low root temperature and aquaporin-mediated root water transport in Arabidopsis.


Assuntos
Aquaporinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Temperatura Baixa , Raízes de Plantas/metabolismo , Aquaporinas/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Transporte Biológico , Membrana Celular/genética , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Cloreto de Mercúrio/farmacologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Brotos de Planta/fisiologia , Estresse Fisiológico , Fatores de Tempo , Água/fisiologia
17.
Plant Cell Physiol ; 52(1): 138-48, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21097474

RESUMO

Functional analysis of a putative novel transcription factor Arabidopsis MYB-like protein designated AtMYBL, which contains two predicted DNA-binding domains, was performed. The physiological role of the R-R-type MYB-like transcription factor has not been reported in any plant. Analyses of an AtMYBL promoter-ß-glucuronidase (GUS) construct revealed substantial gene expression in old leaves and induction of GUS activity by ABA and salt stress. AtMYBL-overexpressing plants displayed a markedly enhanced leaf senescence phenotype. Moreover, the ectopic expression of the AtMYBL gene was very significantly influential in senescence parameters including Chl content, membrane ion leakage and the expression of senescence-related genes. Although the seed germination rate was improved under ABA and saline stress conditions in the AtMYBL-overexpressing plants, decreased salt tolerance was evident compared with the wild type and atmybl RNA interference lines during later seedling growth when exposed to long-term salt stress, indicating that AtMYBL protein is able to developmentally regulate stress sensitivity. Furthermore, AtMYBL protein activated the transcription of a reporter gene in yeast. Green fluorescent protein-tagged AtMYBL was localized in the nuclei of transgenic Arabidopsis cells. Taken together, these results suggest that AtMYBL functions in the leaf senescence process, with the abiotic stress response implicated as a putative potential transcription factor.


Assuntos
Arabidopsis/fisiologia , Genes myb , Folhas de Planta/fisiologia , Sequência de Aminoácidos , Arabidopsis/genética , Glucuronidase/genética , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos
18.
Transgenic Res ; 19(3): 489-97, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19851881

RESUMO

Over the past decade various approaches have been used to increase the expression level of recombinant proteins in plants. One successful approach has been to target proteins to specific subcellular sites/compartments of plant cells, such as the chloroplast. In the study reported here, hyperthermostable endoglucanase Cel5A was targeted into the chloroplasts of tobacco plants via the N-terminal transit peptide of nuclear-encoded plastid proteins. The expression levels of Cel5A transgenic lines were then determined using three distinct transit peptides, namely, the light-harvesting chlorophyll a/b-binding protein (CAB), Rubisco small subunit (RS), and Rubisco activase (RA). RS:Cel5A transgenic lines produced highly stable active enzymes, and the protein accumulation of these transgenic lines was up to 5.2% of the total soluble protein in the crude leaf extract, remaining stable throughout the life cycle of the tobacco plant. Transmission election microscopy analysis showed that efficient targeting of Cel5A protein was under the control of the transit peptide.


Assuntos
Arabidopsis/enzimologia , Celulase/metabolismo , Cloroplastos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Thermotoga maritima/enzimologia , Tabaco/metabolismo , Celulase/ultraestrutura , Primers do DNA/genética , Immunoblotting , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Plasmídeos/genética , Transformação Genética
19.
Physiol Plant ; 135(4): 426-35, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19210750

RESUMO

Recently, we have isolated salt-tolerance genes (SATs) on the basis of the overexpression screening of yeast with a maize cDNA library from kernels. One of the selected genes [salt-tolerance 32 (SAT32)] appears to be a key determinant for salt stress tolerance in yeast cells. Maize SAT32 cDNA encodes for a 49-kDa protein, which is 41% identity with the Arabidopsis salt-tolerance 32 (AtSAT32) unknown gene. Arabidopsis Transfer-DNA (T-DNA) knockout AtSAT32 (atsat32) altered root elongation, including reduced silique length and reduced seed number. In an effort to further assess salinity tolerance in Arabidopsis, we have functionally characterized the AtSAT32 gene and determined that salinity and the plant hormone ABA induced the expression of AtSAT32. The atsat32 mutant was more sensitive to salinity than the wild-type plant. On the contrary, Arabidopsis overexpressing AtSAT32 (35S::AtSAT32) showed enhanced salt tolerance and increased activity of vacuolar H(+)-pyrophosphatase (V-PPase, EC 3.6.1.1) under high-salt conditions. Consistent with these observations, 35S::AtSAT32 plants exhibited increased expression of salt-responsive and ABA-responsive genes, including the Rd29A, Erd15, Rd29B, Rd22 and RAB18 genes. Therefore, our results indicate that AtSAT32 is involved in both salinity tolerance and ABA signaling as a positive regulator in Arabidopsis.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Genes Reguladores , Plantas Tolerantes a Sal , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Membrana Celular/enzimologia , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Pirofosfatase Inorgânica/metabolismo , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Alinhamento de Sequência , Cloreto de Sódio/farmacologia , Vacúolos/enzimologia
20.
Plant Mol Biol ; 64(6): 621-32, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17522953

RESUMO

Despite the high isoform multiplicity of aquaporins in plants, with 35 homologues including 13 plasma membrane intrinsic proteins (PIPs) in Arabidosis thaliana, the individual and integrated functions of aquaporins under various physiological conditions remain unclear. To better understand aquaporin functions in plants under various stress conditions, we examined transgenic Arabidopsis and tobacco plants that constitutively overexpress Arabidopsis PIP1;4 or PIP2;5 under various abiotic stress conditions. No significant differences in growth rates and water transport were found between the transgenic and wild-type plants when grown under favorable growth conditions. The transgenic plants overexpressing PIP1;4 or PIP2;5 displayed a rapid water loss under dehydration stress, which resulted in retarded germination and seedling growth under drought stress. In contrast, the transgenic plants overexpressing PIP1;4 or PIP2;5 showed enhanced water flow and facilitated germination under cold stress. The expression of several PIPs was noticeably affected by the overexpression of PIP1;4 or PIP2;5 in Arabidopsis under dehydration stress, suggesting that the expression of one aquaporin isoform influences the expression levels of other aquaporins under stress conditions. Taken together, our results demonstrate that overexpression of an aquaporin affects the expression of endogenous aquaporin genes and thereby impacts on seed germination, seedling growth, and stress responses of the plants under various stress conditions.


Assuntos
Aquaporinas/genética , Aquaporinas/metabolismo , Arabidopsis/genética , Plantas Geneticamente Modificadas , Tabaco/genética , Membrana Celular/metabolismo , Clorofila/química , Temperatura Baixa , Meio Ambiente , Genes de Plantas , Vetores Genéticos , Microscopia de Fluorescência , Fenótipo , Plantas Geneticamente Modificadas/genética , Pressão , Fatores de Tempo , Água/química , Água/metabolismo
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