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1.
Artigo em Inglês | MEDLINE | ID: mdl-32423827

RESUMO

The reactive oxygen species (ROS) are continuously produced and are essential for mediating the growth and development of plants. However too much accumulation of ROS can result in the oxidative damage to cells, especially under the adverse environmental conditions. Plants have evolved sophisticated strategies to regulate the homeostasis of H2O2. In this study, we generated transgenic Arabidopsis plants in the Ws ecotype (Ws) background in which WRKY33 is co-suppressed (csWRKY33/Ws). Compared with Ws, csWRKY33/Ws plants accumulate more H2O2. RNA-seq analysis indicated that in csWRKY33/Ws plants, expression of oxidative stress related genes such as ascorbate peroxidase 2 (APX2) is affected. Over-expression of APX2 can rescue the phenotype of csWRKY33/Ws, suggesting that the changes in the growth of csWRKY33/Ws is duo to the higher accumulation of H2O2. Analysis of the CHIP-seq data suggested that WRKY33 can directly regulate the expression of PIF4, vice versa. qPCR analysis also confirmed that the mutual regulation between WRKY33 and PIF4. Similar to that of csWRKY33/Ws, and the accumulation of H2O2 in pif4 also increased. Taken together, our results reveal a WRKY33-PIF4 regulatory loop that appears to play an important role in regulating the growth and development of seedlings by mediating H2O2 homeostasis.

2.
J Agric Food Chem ; 2020 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-32396374

RESUMO

Genetic engineering (GE) technology is widely used in plant modification. However, the results of modification may not exactly meet the expectations. Herein we propose a new multi-omics method for GE plant evaluation based on the optimized use of the metID algorithm. Using this method, we found that flavonoids accumulation was at the expense of the great sacrifice of L-phenylalanine in GE tomatoes for the first time. Meanwhile, the ceramide series of sphingolipid is synthesized de novo from L-serine, and ceramides are the primary source of vesicles which coated with flavonoids and secreted from the endoplasmic reticulum. Therefore, the accumulation of ceramide series of sphingolipid changed the cell component of intracellular organelles. Furthermore, the improvement of the method allows us to identify more metabolites related to dysregulated pathways.

3.
Biomolecules ; 10(4)2020 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-32295207

RESUMO

Previous investigations have shown that the SUPPRESSORS OF MAX2 1-LIKE6, 7 and 8 (SMXL6, 7 and 8) proteins redundantly repress strigolactone (SL) signaling in plant growth and development. Recently, a growing body of evidence indicated that SLs positively regulate plant drought resistance through functional analyses of genes involved in SL biosynthesis and positive regulation of SL signaling. However, the functions of the SL-signaling negative regulators SMXL6, 7 and 8 in drought resistance and the associated mechanisms remain elusive. To reveal the functions of these SMXL proteins, we analyzed the drought-resistant phenotype of the triple smxl6,7,8 mutant plants and studied several drought resistance-related traits. Our results showed that the smxl6,7,8 mutant plants were more resistant to drought than wild-type plants. Physiological investigations indicated that the smxl6,7,8 mutant plants exhibited higher leaf surface temperature, reduced cuticle permeability, as well as decreases in drought-induced water loss and cell membrane damage in comparison with wild-type plants. Additionally, smxl6,7,8 mutant plants displayed an increase in anthocyanin biosynthesis during drought, enhanced detoxification capacity and increased sensitivity to abscisic acid in cotyledon opening and growth inhibition assays. A good correlation between the expression levels of some relevant genes and the examined physiological and biochemical traits was observed. Our findings together indicate that the SMXL6, 7 and 8 act as negative regulators of drought resistance, and that disruption of these SMXL genes in crops may provide a novel way to improve their drought resistance.

4.
Chem Biodivers ; 17(2): e1900473, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31961474

RESUMO

Veratrum plant contains a family of compounds called steroidal alkaloids which have been previously reported to cause DNA damage and blood pressure decrease in vivo. In this study, the antihypertensive effects and DNA damage in brain cells of 12 steroidal alkaloids separated from Veratrum plant were all evaluated to develop a relationship among chemical structure, antihypertensive activity and neurotoxicity by utilization of chemical principal component analysis (PCA) and hierarchical cluster analysis (HCA). Twelve steroidal alkaloids markedly reduced high blood pressure of hypertensive mice and also similarly induced varying degrees of DNA single-strand breaks in mouse cerebellum and cerebral cortex after oral administration. On the basis of the PCA and HCA results, it was suggested that the 3-carboxylic esters and benzene group play a core role in the DNA damage of brain cells, while more hydroxy groups in the A-ring and B-ring structure of jervine-type alkaloid led to stronger antihypertensive activity. The primary structure, activity and neurotoxicity relationship were discussed briefly.


Assuntos
Anti-Hipertensivos/química , Alcaloides de Veratrum/química , Veratrum/química , Administração Oral , Animais , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Análise por Conglomerados , Dano ao DNA/efeitos dos fármacos , Camundongos , Extratos Vegetais/química , Análise de Componente Principal , Relação Estrutura-Atividade , Veratrum/metabolismo , Alcaloides de Veratrum/farmacologia
5.
Biochem Biophys Res Commun ; 521(1): 184-189, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31630799

RESUMO

In order to withstand high light (HL) stress, plants have evolved both short-term defense and repair mechanisms and long-term acclimation responses. At present, however, the underlying signaling events and molecular mechanisms are still poorly understood. Analysis of the mutants coe1, coe1 gun1 double mutant and oeGUN1coe1 revealed increased sensitivity to HL stress as compared to wild type (WT), with oeGUN1 coe1 plants displaying the highest sensitivity. Accumulation of FTSH2 protein and degradation of D1 protein during the HL stress were shown to depend on both COE1 and GUN1. Overexpression of COE1 enhanced the induction of FTSH2 and the tolerance to HL stress. These results indicate that the COE1-GUN1 signaling pathway plays an important role in regulating the adaptation of plants to HL.

6.
Evol Bioinform Online ; 15: 1176934319889948, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31798299

RESUMO

Determining the genetic rearrangement and domestication footprints in Gossypium hirsutum cultivars and primitive race genotypes are essential for effective gene conservation efforts and the development of advanced breeding molecular markers for marker-assisted breeding. In this study, 94 accessions representing the 7 primitive races of G hirsutum, along with 9 G hirsutum and 12 Gossypium barbadense cultivated accessions were evaluated. The genotyping-by-sequencing (GBS) approach was employed and 146 558 single nucleotide polymorphisms (SNP) were generated. Distinct SNP signatures were identified through the combination of selection scans and association analyses. Phylogenetic analyses were also conducted, and we concluded that the Latifolium, Richmondi, and Marie-Galante race accessions were more genetically related to the G hirsutum cultivars and tend to cluster together. Fifty-four outlier SNP loci were identified by selection-scan analysis, and 3 SNPs were located in genes related to the processes of plant responding to stress conditions and confirmed through further genome-wide signals of marker-phenotype association analysis, which indicate a clear selection signature for such trait. These results identified useful candidate gene locus for cotton breeding programs.

7.
Biochem Biophys Res Commun ; 520(2): 366-372, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31606202

RESUMO

As a scaffold protein, Receptor for Activated C Kinase 1a (RACK1) interacts with many proteins and is involved in multiple biological processes in Arabidopsis. However, the global RACK1 protein interaction network in higher plants remains poorly understood. Here, we generated a yeast two-hybrid library using mixed samples from different developmental stages of Arabidopsis thaliana. Using RACK1a as bait, we performed a comprehensive screening of the resulting library to identify RACK1a interactors at the whole-transcriptome level. We selected 1065 independent positive clones that led to the identification of 215 RACK1a interactors. We classified these interactors into six groups according to their potential functions. Several interactors were selected for bimolecular fluorescence complementation (BiFC) analysis and their interaction with RACK1a was confirmed in vivo. Our results provide further insight into the molecular mechanisms through which RACK1a regulates various growth and development processes in higher plants.

8.
J Biotechnol ; 306: 97-104, 2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31550488

RESUMO

Carboxylic acid reductases (CARs) play crucial roles in the biosynthesis of optically pure aldehydes with no side products. It has inspired synthetic organic chemists and biotechnologists to exploit them as catalysts in practical applications. However, levels of activity and substrate specificity are not routinely sufficient. Recent developments in protein engineering have produced numerous biocatalysts with new catalytic properties, whereas such efforts in CARs are limited. In this study, we show that the exploitation of information derived from catalytic mechanism analysis and molecular dynamics simulations assisted the semi-rational engineering of a CAR from Segniliparus rugosus (SrCAR) with the aim of increasing activity. Guided by protein-ligand interaction fingerprinting analysis, 17 residues at the substrate binding pockets were first identified. We then performed single site saturation mutagenesis and successfully obtained variants that gave high activities using benzoic acid as the model substrate. As a result, the best mutant K524W enabled 99% conversion and 17.28 s-1 mM-1kcat/Km, with 7- and 2-fold improvement compared to the wild-type, respectively. The engineered catalyst K524W as well as a second variant K524Q proved to be effective in the reduction of other benzoic acid derivatives. Insight into the source of enhanced activity was gained by molecular dynamics simulations.

9.
Plant Methods ; 14: 50, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29977323

RESUMO

Background: Genetically modified cotton accounts for 64% of the world's cotton growing area (22.3 million hectares). The genome sequencing of the diploid cotton progenitors Gossypium raimondii and Gossypium arboreum as well as the cultivated Gossypium hirsutum has provided a wealth of genetic information that could be exploited for crop improvement. Unfortunately, gene functional characterization in cotton is lagging behind other economically important crops due to the low efficiency, lengthiness and technical complexity of the available stable transformation methods. We present here a simple, fast and efficient method for the transient transformation of G. hirsutum that can be used for gene characterization studies. Results: We developed a transient transformation system for gene characterization in upland cotton. Using ß-glucuronidase as a reporter for Agrobacterium-mediated transformation assays, we evaluated multiple transformation parameters such as Agrobacterium strain, bacterial density, length of co-cultivation, chemicals and surfactants, which can affect transformation efficiency. After the initial characterization, the Agrobacterium EHA105 strain was selected and a number of binary constructs used to perform gene characterization studies. 7-days-old cotton seedlings were co-cultivated with Agrobacterium and transient gene expression was observed 5 days after infection of the plants. Transcript levels of two different transgenes under the control of the cauliflower mosaic virus (CaMV) 35S promoter were quantified by real-time reverse transcription PCR (qRT-PCR) showing a 3-10 times increase over the levels observed in non-infected controls. The expression patterns driven by the promoters of two G. hirsutum genes as well as the subcellular localization of their corresponding proteins were studied using the new transient expression system and our observations were consistent with previously published results using Arabidopsis as a heterologous system. Conclusions: The Agrobacterium-mediated transient transformation method is a fast and easy transient expression system enabling high transient expression and transformation efficiency in upland cotton seedlings. Our method can be used for gene functional studies such as promoter characterization and protein subcellular localization in cotton, obviating the need to perform such studies in a heterologous system such as Arabidopsis.

10.
Plant Methods ; 14: 40, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29872452

RESUMO

Background: The CRISPR/Cas9 system is being used for genome editing purposes by many research groups in multiple plant species. Traditional sequencing methods to identify homozygous mutants are time-consuming, laborious and expensive. Results: We have developed a method to screen CRISPR/Cas9-induced mutants through Mutation Sites Based Specific Primers Polymerase Chain Reaction (MSBSP-PCR). The MSBSP-PCR method was successfully used to identify homozygous/biallelic mutants in Nicotiana tabacum and Arabidopsis thaliana, and we speculate that it can be used for the identification of CRISPR/Cas9-induced mutants in other plant species. Compared to traditional sequencing methods, MSBSP-PCR is simpler, faster and cheaper. Conclusions: The MSBSP-PCR method is simple to implement and can save time and cost in the screening of CRISPR/Cas9-induced homozygous/biallelic mutants.

11.
BMC Plant Biol ; 17(1): 99, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28587634

RESUMO

BACKGROUND: Sinapic acid and its esters have broad functions in different stages of seed germination and plant development and are thought to play a role in protecting against ultraviolet irradiation. To better understand the interactions between sinapic acid esters and seed germination processes in response to various stresses, we analyzed the role of the plant hormone abscisic acid (ABA) in the regulation of sinapic acid esters involved in seed germination and early seedling growth. RESULTS: We found that exogenous sinapic acid promotes seed germination in a dose-dependent manner in Arabidopsis thaliana. High-performance liquid chromatography mass spectrometry analysis showed that exogenous sinapic acid increased the sinapoylcholine content of imbibed seeds. Furthermore, sinapic acid affected ABA catabolism, resulting in reduced ABA levels and increased levels of the ABA-glucose ester. Using mutants deficient in the synthesis of sinapate esters, we showed that the germination of mutant sinapoylglucose accumulator 2 (sng2) and bright trichomes 1 (brt1) seeds was more sensitive to ABA than the wild-type. Moreover, Arabidopsis mutants deficient in either abscisic acid deficient 2 (ABA2) or abscisic acid insensitive 3 (ABI3) displayed increased expression of the sinapoylglucose:choline sinapoyltransferase (SCT) and sinapoylcholine esterase (SCE) genes with sinapic acid treatment. This treatment also affected the accumulation of sinapoylcholine and free choline during seed germination. CONCLUSIONS: We demonstrated that sinapoylcholine, which constitutes the major phenolic component in seeds among various minor sinapate esters, affected ABA homeostasis during seed germination and early seedling growth in Arabidopsis. Our findings provide insights into the role of sinapic acid and its esters in regulating ABA-mediated inhibition of Arabidopsis seed germination in response to drought stress.


Assuntos
Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Ácidos Cumáricos/metabolismo , Germinação , Reguladores de Crescimento de Planta/metabolismo , Ácido Abscísico/antagonistas & inibidores , Arabidopsis/embriologia , Arabidopsis/genética , Ésteres/metabolismo , Homeostase , Mutação , Plântula/crescimento & desenvolvimento
12.
Int J Mol Sci ; 16(12): 30438-57, 2015 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-26703579

RESUMO

Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene ß cyclase (ß-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a ß-LCY gene from Nicotiana tabacum, designated as Ntß-LCY1, was cloned and functionally characterized. Robust expression of Ntß-LCY1 was found in leaves, and Ntß-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntß-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntß-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntß-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum.


Assuntos
Secas , Liases Intramoleculares/genética , Proteínas de Plantas/genética , Tolerância ao Sal/genética , Tabaco/genética , Ácido Abscísico/metabolismo , Sequência de Aminoácidos , Liases Intramoleculares/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Tabaco/enzimologia , Tabaco/metabolismo
13.
Yao Xue Xue Bao ; 50(3): 337-9, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26118114

RESUMO

To study the chemical constituents of Veratrum dahuricum (Turcz.) Loes. f., a new aurone glycoside named as (Z)-7, 4'-dimethoxy-6-hydroxyl-aurone-4-O-ß-glucopyranoside was isolated from the 95% ethanol extracts of the rhizomes and roots of Veratrum dahuricum (Turcz.) Loes. f. by repeated column chromatography on silica gel and recrystallization. Its structure was established by extensive spectroscopic analyses, and its cytotoxicities against HepG-2, MCF7 and A549 cell lines were measured in vitro.


Assuntos
Benzofuranos/isolamento & purificação , Glicosídeos/isolamento & purificação , Veratrum/química , Linhagem Celular Tumoral , Humanos , Raízes de Plantas/química , Plantas Medicinais/química , Rizoma/química
14.
J Chromatogr Sci ; 53(7): 1092-9, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25547283

RESUMO

Veratramine (VAM) is the major lipid-soluble alkaloid existing in Veratrum nigrum L. that has been demonstrated to exert neurotoxic effects. To better understand the potential mechanism of neurotoxicity of VAM, VAM-induced DNA damage was measured in the cerebellum and cerebral cortex of mice after a 7-day repetitive oral dose by using single-cell gel electrophoresis (comet assay). A method based on high-performance liquid chromatography-electrospray ionization tandem mass spectrometry was developed for the determination of VAM and its in vivo and in vitro metabolites, to establish the potential correlation between metabolites and neurotoxicity. In vitro experiment was carried out using rat liver microsomes, whereas the in vivo study was conducted on rats at a single dose of 3 mg/kg. The results showed that VAM caused DNA damage in the cerebellum and cerebral cortex of mice in a dose-dependent manner. Phenyl mono-oxidation, sulfate conjugation and phenyl di-oxidation were proposed to be the main in vivo metabolic pathways of VAM, whereas the major in vitro metabolic pathways were phenyl mono-oxidation, hydroxylation and methylation. Phenyl-oxidation reaction was likely to be associated with reactive oxygen species production, leading to the DNA damage in the mouse brain.


Assuntos
Encéfalo/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Alcaloides de Veratrum/metabolismo , Alcaloides de Veratrum/toxicidade , Animais , Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Ratos , Espectrometria de Massas por Ionização por Electrospray/métodos , Veratrum/química , Alcaloides de Veratrum/administração & dosagem , Alcaloides de Veratrum/isolamento & purificação
15.
Yao Xue Xue Bao ; 46(11): 1332-7, 2011 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-22260024

RESUMO

This paper is to report the study of resveratrol-induced apoptosis and its mechanisms in MCF-7 cells. MTT assay was performed to assess the cytotoxicity of resveratrol on MCF-7 cells. Hoechst 33258 staining was used to observe cellular morphologic changes in apoptosis. Apoptosis was measured by flow cytometric analysis and the protein expression was examined by Western blotting analysis. The results indicated that resveratrol could inhibit MCF-7 cell growth in a time- and concentration-dependent manner. Remarkable morphologic changes in the cells after 60 micromol L(-1) resveratrol treatment, including cell nuclear shrinkage, DNA condensation and apoptotic bodies, were observed by Hoechst 33258 staining. Resveratrol could induce apoptosis and activate p38 and p53 in a time dependent manner in MCF-7 cells. In addition, the cell growth inhibitory ratio and the apoptotic ratio of resveratrol-treated group decreased markedly by the p38 MAPK inhibitor SB203580 or p53 inhibitor pifithrin-alpha. Further experiments confirmed that resveratrol-induced p53 activation was reduced by SB203580 whereas the activation of p38 was not affected by pifithrin-alpha. In conclusion, resveratrol induced apoptosis in MCF-7 cells could be through activating p38-p53 signal pathway.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Transdução de Sinais , Estilbenos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Antineoplásicos Fitogênicos/administração & dosagem , Benzotiazóis/farmacologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Humanos , Imidazóis/farmacologia , Células MCF-7 , Piridinas/farmacologia , Resveratrol , Estilbenos/administração & dosagem , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
16.
Zhongguo Zhong Yao Za Zhi ; 34(14): 1816-8, 2009 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-19894515

RESUMO

The chemical constituents of Leonurus heterophyllus were separated and purified by repeated column chromatography on silica gel, HPD 100, Sephadex LH-20, and PHPLC. Each compound was characterized by spectroscopic and physical data. Eight compounds have been purified and identified to be quercetin 3-O-robinobioside (1), rutin (2), isoquerci trin (3), hyperoside (4), quercetin (5), apigenin (6), genkwanin (7), and benzoic acid (8). Among them, compounds 2, 5-7 were isolated from L. heterophyllus for the first time; Compounds 1, 3, 4, 8 were obtained for the first time from the genus Leonurus. The in vitro activities against leukemia K562 Cells of pure components were evaluated by testing their IC50. Compounds 1-6, 8 exhibited in-vitro inhibitory activities against leukemia K562 cells in different extent.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Leonurus/química , Antineoplásicos/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Medicamentos de Ervas Chinesas/isolamento & purificação , Humanos , Células K562
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