RESUMO
Drought and salt stress severely inhibit plant growth and development; however, the regulatory mechanisms of plants in response to these stresses are not fully understood. Here we report that the expression of a WRKY transcription factor WRKY46 is rapidly induced by drought, salt and oxidative stresses. T-DNA insertion of WRKY46 leads to more sensitivity to drought and salt stress, whereas overexpression of WRKY46 (OV46) results in hypersensitivity in soil-grown plants, with a higher water loss rate, but with increased tolerance on the sealed agar plates. Stomatal closing in the OV46 line is insensitive to ABA because of a reduced accumulation of reactive oxygen species (ROS) in the guard cells. We further find that WRKY46 is expressed in guard cells, where its expression is not affected by dehydration, and is involved in light-dependent stomatal opening. Microarray analysis reveals that WRKY46 regulates a set of genes involved in cellular osmoprotection and redox homeostasis under dehydration stress, which is confirmed by ROS and malondialdehyde (MDA) levels in stressed seedlings. Moreover, WRKY46 modulates light-dependent starch metabolism in guard cells via regulating QUA-QUINE STARCH (QQS) gene expression. Taken together, we demonstrate that WRKY46 plays dual roles in regulating plant responses to drought and salt stress and light-dependent stomatal opening in guard cells.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Secas , Perfilação da Expressão Gênica , Homeostase , Luz , Malondialdeído/metabolismo , Mutagênese Insercional , Análise de Sequência com Séries de Oligonucleotídeos , Pressão Osmótica , Oxirredução , Estresse Oxidativo , Fenótipo , Estômatos de Plantas/citologia , Estômatos de Plantas/genética , Estômatos de Plantas/fisiologia , Estômatos de Plantas/efeitos da radiação , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio/metabolismo , Sais , Plântula/citologia , Plântula/genética , Plântula/fisiologia , Plântula/efeitos da radiação , Amido/metabolismo , Fatores de Transcrição/genéticaRESUMO
The transcription factor WRKY family is one type of key regulatory components of plant development and defense against stress factors. The expression profiles of three AtWRKY genes under abiotic stresses were analyzed by Northern blotting analysis. The expression of AtWRKY25, AtWRKY26, and AtWRKY33 changed during stress treatments including thermal factors, NaCl, abscisic acid (ABA) and osmotic stress, and significantly under NaCl and cold treatments, suggesting a specific role of the three AtWRKYs in adaptation to environmental stresses in plants. We also found that the three AtWRKY genes showed distinct expression patterns under thermal stresses. AtWRKY25 and AtWRKY26 were gradually induced during heat and cold treatments, whereas AtWRKY33 was suppressed by heat treatment and induced rapidly during cold stress, indicating that the three AtWRKYs may play different roles in response to temperature factors. In addition, we analyzed the sequence of the promoters with bioinformatics approach, and some cis-elements involved in abiotic stresses and hormonal responses were revealed. The results provided important information for studying biological functions of three AtWRKY genes.
Assuntos
Proteínas de Arabidopsis/genética , Perfilação da Expressão Gênica , Fatores de Transcrição/genética , Ácido Abscísico/farmacologia , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Northern Blotting , Biologia Computacional , Dados de Sequência Molecular , Cloreto de Sódio/farmacologia , Temperatura , Fatores de Transcrição/químicaRESUMO
Two predicted peptide genes in Arabidopsis thaliana L., Peptide5 and Peptide6, was confirmed by RT-PCR in mRNA level. The expression profile indicated that both genes were generally expressed at different developmental stages and tissues as constitutive gene expression, and they also responded to six treatments including NaCl, PEG, MeJA (methyl jasmonate), SA (salicylic acid), cold and wound in transcription level. Analysis of the promoter sequence suggests that Peptide5 in Arabidopsis may contribute to the secondary xylem formation.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Peptídeos/genética , Acetatos/farmacologia , Arabidopsis/efeitos dos fármacos , Sequência de Bases , Temperatura Baixa , Ciclopentanos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Oxilipinas/farmacologia , Polietilenoglicóis/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido Salicílico/farmacologia , Cloreto de Sódio/farmacologiaRESUMO
OBJECTIVE: To construct the plant expression vector containing the nucleotide sequence encoding cholera toxin B (CTB) subunits. METHOD: Using high-fidelity PCR, we amplified CTB genes that were then subcloned into the transition vector pRTL2. Following confirmation of the CTB nucleotide sequence, the vector was subcloned into the plant vector pBI121 that was subsequently transferred into Agrobacterium tumefaciens LBA4404 by electroporation. RESULTS: CTB DNA that was ligated into the transition vectors resulted in the 2 vectors designated as pRCTB and pRCTBK. After the 2 vectors were ligated into the plant binary vector pBI121 respectively, new plant binary vectors, namely pBI-CTB and pBI-CTBK, were produced. Analysis with restriction endonucleases confirmed successful transfer of pBI-CTB and pBI-CTBK into Agrobacterium tumefaciens LBA4404. CONCLUSION: With appropriate technological strategy, the plant binary expression vectors encoding CTB have been constructed, which facilitates further investigation of CTB protein expressions in transgenic plant.
Assuntos
Toxina da Cólera/genética , Genes de Plantas , Toxina da Cólera/biossíntese , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/genéticaRESUMO
It is well known that the type III secretion system (T3SS) and type III (T3) effectors are essential for the pathogenicity of most bacterial phytopathogens and that the expression of T3SS and T3 effectors is suppressed in rich media but induced in minimal media and plants. To facilitate in-depth studies on T3SS and T3 effectors, it is crucial to establish a medium for T3 effector expression and secretion. Xanthomonas campestris pv. campestris (Xcc) is a model bacterium for studying plant-pathogen interactions. To date no medium for Xcc T3 effector secretion has been defined. Here, we compared four minimal media (MME, MMX, XVM2, and XOM2) which are reported for T3 expression induction in Xanthomonas spp. and found that MME is most efficient for expression and secretion of Xcc T3 effectors. By optimization of carbon and nitrogen sources and pH value based on MME, we established XCM1 medium, which is about 3 times stronger than MME for Xcc T3 effectors secretion. We further optimized the concentration of phosphate, calcium, and magnesium in XCM1 and found that XCM1 with a lower concentration of magnesium (renamed as XCM2) is about 10 times as efficient as XCM1 (meanwhile, about 30 times stronger than MME). Thus, we established an inducing medium XCM2 which is preferred for T3 effector secretion in Xcc.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Meios de Cultura/química , Fatores de Virulência/metabolismo , Xanthomonas campestris/crescimento & desenvolvimento , Xanthomonas campestris/metabolismoRESUMO
It is well known that the type III secretion system (T3SS) and type III (T3) effectors are essential for the pathogenicity of most bacterial phytopathogens and that the expression of T3SS and T3 effectors is suppressed in rich media but induced in minimal media and plants. To facilitate in-depth studies on T3SS and T3 effectors, it is crucial to establish a medium for T3 effector expression and secretion. Xanthomonas campestris pv. campestris (Xcc) is a model bacterium for studying plant-pathogen interactions. To date no medium for Xcc T3 effector secretion has been defined. Here, we compared four minimal media (MME, MMX, XVM2, and XOM2) which are reported for T3 expression induction in Xanthomonas spp. and found that MME is most efficient for expression and secretion of Xcc T3 effectors. By optimization of carbon and nitrogen sources and pH value based on MME, we established XCM1 medium, which is about 3 times stronger than MME for Xcc T3 effectors secretion. We further optimized the concentration of phosphate, calcium, and magnesium in XCM1 and found that XCM1 with a lower concentration of magnesium (renamed as XCM2) is about 10 times as efficient as XCM1 (meanwhile, about 30 times stronger than MME). Thus, we established an inducing medium XCM2 which is preferred for T3 effector secretion in Xcc.
Assuntos
Receptores dos Hormônios Tireóideos , Western Blotting , Xanthomonas campestris , Glucuronidase , Tri-IodotironinaRESUMO
It is well known that the type III secretion system (T3SS) and type III (T3) effectors are essential for the pathogenicity of most bacterial phytopathogens and that the expression of T3SS and T3 effectors is suppressed in rich media but induced in minimal media and plants. To facilitate in-depth studies on T3SS and T3 effectors, it is crucial to establish a medium for T3 effector expression and secretion. Xanthomonas campestris pv. campestris (Xcc) is a model bacterium for studying plant-pathogen interactions. To date no medium for Xcc T3 effector secretion has been defined. Here, we compared four minimal media (MME, MMX, XVM2, and XOM2) which are reported for T3 expression induction in Xanthomonas spp. and found that MME is most efficient for expression and secretion of Xcc T3 effectors. By optimization of carbon and nitrogen sources and pH value based on MME, we established XCM1 medium, which is about 3 times stronger than MME for Xcc T3 effectors secretion. We further optimized the concentration of phosphate, calcium, and magnesium in XCM1 and found that XCM1 with a lower concentration of magnesium (renamed as XCM2) is about 10 times as efficient as XCM1 (meanwhile, about 30 times stronger than MME). Thus, we established an inducing medium XCM2 which is preferred for T3 effector secretion in Xcc.