RESUMO
Mildew resistance locus o (MLO) proteins are heptahelical integral membrane proteins of which some isoforms act as susceptibility factors for the powdery mildew pathogen. In many angiosperm plant species, loss-of-function mlo mutants confer durable broad-spectrum resistance against the fungal disease. Barley Mlo is known to interact via a cytosolic carboxyl-terminal domain with the intracellular calcium sensor calmodulin (CAM) in a calcium-dependent manner. Site-directed mutagenesis has revealed key amino acid residues in the barley Mlo calmodulin-binding domain (CAMBD) that, when mutated, affect the MLO-CAM association. We here tested the respective interaction between Arabidopsis thaliana MLO2 and CAM2 using seven different types of in vitro and in vivo protein-protein interaction assays. In each assay, we deployed a wild-type version of either the MLO2 carboxyl terminus (MLO2CT), harboring the CAMBD, or the MLO2 full-length protein and corresponding mutant variants in which two key residues within the CAMBD were substituted by non-functional amino acids. We focused in particular on the substitution of two hydrophobic amino acids (LW/RR mutant) and found in most protein-protein interaction experiments reduced binding of CAM2 to the corresponding MLO2/MLO2CT-LW/RR mutant variants in comparison with the respective wild-type versions. However, the Ura3-based yeast split-ubiquitin system and in planta bimolecular fluorescence complementation (BiFC) assays failed to indicate reduced CAM2 binding to the mutated CAMBD. Our data shed further light on the interaction of MLO and CAM proteins and provide a comprehensive comparative assessment of different types of protein-protein interaction assays with wild-type and mutant versions of an integral membrane protein.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Calmodulina , Domínios e Motivos de Interação entre Proteínas , Arabidopsis/genética , Arabidopsis/metabolismo , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Arabidopsis/metabolismo , Mapeamento de Interação de Proteínas/métodosRESUMO
PURPOSE: Purple acid phosphatases (PAPs) includ the largest classes of non-specific plant acid phosphatases. Most characterized PAPs were found to play physiological functions in phosphorus metabolism. In this study, we investigated the function of AtPAP17 gene encoding an important purple acid phosphatase in Arabidopsis thaliana. METHODS: The full-length cDNA sequence of AtPAP17 gene under the control of CaMV-35S promoter was transferred to the A. thaliana WT plant. The generated homozygote AtPAP17-overexpressed plants were compared by the types of analyses with corresponding homozygote atpap17-mutant plant and WT in both + P (1.2 mM) and - P (0 mM) conditions. RESULTS: In the + P condition, the highest and the lowest amount of Pi was observed in AtPAP17-overexpressed plants and atpap17-mutant plants by 111% increase and 38% decrease compared with the WT plants, respectively. Furthermore, under the same condition, APase activity of AtPAP17-overexpressed plants increased by 24% compared to the WT. Inversely, atpap17-mutant plant represented a 71% fall compared to WT plants. The comparison of fresh weight and dry weight in the studied plants showed that the highest and the lowest amount of absorbed water belonged to OE plants (with 38 and 12 mg plant-1) and Mu plants (with 22 and 7 mg plant-1) in + P and - P conditions, respectively. CONCLUSION: The lack of AtPAP17 gene in the A. thaliana genome led to a remarkable reduction in the development of root biomass. Thus, AtPAP17 could have an important role in the root but not shoot developmental and structural programming. Consequently, this function enables them to absorb more water and eventually associated with more phosphate absorption.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Fósforo , Glicoproteínas/genética , Fosfatase Ácida/genética , Fosfatase Ácida/química , Fosfatase Ácida/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fosfatos , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/genética , Raízes de Plantas/metabolismoRESUMO
How likely genetic variations associated with environment identified in silico from genome wide association study are functionally relevant to environmental adaptation has been largely unexplored experimentally. Here we analyzed top 29 genes containing polymorphisms associated with local temperature variation (minimum, mean, maximum) among 1129 natural accessions of Arabidopsis thaliana. Their loss-of-function mutants were assessed for growth and stress tolerance at five temperatures. Twenty genes were found to affect growth or tolerance at one or more of these temperatures. Significantly, genes associated with maximum temperature more likely have a detect a function at higher temperature, while genes associated with minimum temperature more likely have a function at lower temperature. In addition, gene variants are distributed more frequently at geographic locations where they apparently offer an enhanced growth or tolerance for five genes tested. Furthermore, variations in a large proportion of the in silico identified genes associated with minimum or mean-temperatures exhibited a significant association with growth phenotypes experimentally assessed at low temperature for a small set of natural accessions. This study shows a functional relevance of gene variants associated with environmental variables and supports the feasibility of the use of local temperature factors in investigating the genetic basis of temperature adaptation.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Estudo de Associação Genômica Ampla , TemperaturaRESUMO
Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of a target gene encoding modified human interferon (dIFN) using endonuclease Cas9. For the targeted insertion, we selected the region of the histone H3.3 gene (HTR5) with a high constitutive level of expression. Our results indicated that Cas9-induced DNA integration occurred with the highest frequency with the construction with donor DNA surrounded by homology arms and Cas9 endonuclease recognition sites. Among the monoclones of the four cell lines with knock-in studied, there is high heterogeneity in the level of expression and accumulation of the target protein. The accumulation of dIFN protein in cell lines with targeted insertions into the target region of the HTR5 gene does not statistically differ from the level of accumulation of dIFN protein in the group of lines with random integration of the transgene. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Histonas/metabolismo , Técnicas de Cultura de CélulasRESUMO
With widespread applications of the latest neonicotinoid in agriculture, dinotefuran has gradually become a hazardous contaminant for plants through the generation of excessive reactive oxygen species. However, the potential toxic mechanisms of oxidative damages to plants induced by dinotefuran are still unknown. As a core component of the glutathione antioxidant enzyme system, glutathione peroxidases have been used as biomarkers to reflect excessive oxidative stress. In this study, the hazardous effects of dinotefuran on AtGPX6 were investigated at the molecular level. The intrinsic fluorescence intensity of AtGPX6 was quenched using the static quenching mechanism upon binding with dinotefuran. Moreover, a single binding site was predicted for AtGPX6 toward dinotefuran, and the complex formation was presumed to be driven by hydrogen bonds or van der Waals forces, which conformed with the molecular docking results. In addition, AtGPX6 exhibited moderate binding affinity with dinotefuran based on the bio-layer interferometry assay. In addition, the loosening and unfolding of the protein skeleton of AtGPX6 with the addition of dinotefuran were explored along with the increase of hydrophobicity around tryptophan residues. Lastly, the toxic effects of dinotefuran on the root growth of Arabidopsis seedlings were also examined. The exploration of the binding mechanism of dinotefuran with AtGPX6 at the molecular level would provide the toxicity assessment of dinotefuran on plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Guanidinas/farmacologia , Inseticidas/farmacologia , Neonicotinoides/farmacologia , Nitrocompostos/farmacologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação , Inseticidas/química , Simulação de Acoplamento Molecular , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/química , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Plântula/química , Plântula/efeitos dos fármacos , Plântula/enzimologia , Plântula/genéticaRESUMO
ATR, a DNA damage signaling kinase, is required for cell cycle checkpoint regulation and detecting DNA damage caused by genotoxic factors including Al3+ ions. We analyzed the function of the HvATR gene in response to chemical clastogen-maleic acid hydrazide (MH). For this purpose, the Al-tolerant barley TILLING mutant hvatr.g was used. We described the effects of MH on the nuclear genome of hvatr.g mutant and its WT parent cv. "Sebastian", showing that the genotoxic effect measured by TUNEL test and frequency of cells with micronuclei was much stronger in hvatr.g than in WT. MH caused a significant decrease in the mitotic activity of root cells in both genotypes, however this effect was significantly stronger in "Sebastian". The impact of MH on the roots cell cycle, analyzed using flow cytometry, showed no differences between the mutant and WT.
Assuntos
Alumínio/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA/efeitos dos fármacos , Hordeum/efeitos dos fármacos , Hidrazida Maleica/farmacologia , Proteínas de Arabidopsis/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Dano ao DNA/genética , Genoma de Planta/efeitos dos fármacos , Genoma de Planta/genética , Genótipo , Hordeum/genética , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Mutagênicos/farmacologia , Mutação/efeitos dos fármacos , Mutação/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genéticaRESUMO
For an extensive period of time apical meristem (SAM) has been considered as a mysterious organ, due to its small, hidden and dynamic structure. Confocal imaging, combined with fluorescent reporters, enables researchers to unveil the mechanisms underlying cellular activities, such as gene expression, cell division, growth patterns and cell-cell communications. Recently, a series of protocols were developed for confocal imaging of inflorescence meristem (IM) and floral meristem (FM). However, the requirement of high configuration, such as the need of a water-dipping lens without coverslip and the specialized turrets associated with fixed-stage microscopes, impedes the wide adoption of these methods. We exploited an improved object slide and matching method aiming to decrease the configuration requirement. Following this protocol, various dry microscope lenses can be selected with flexibility for building 3D images of IM and FM.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/fisiologia , Meristema/crescimento & desenvolvimento , Arabidopsis/citologia , Flores/citologia , Meristema/citologia , Microscopia ConfocalRESUMO
Plants balance their competing requirements for growth and stress tolerance via a sophisticated regulatory circuitry that controls responses to the external environments. We have identified a plant-specific gene, COST1 (constitutively stressed 1), that is required for normal plant growth but negatively regulates drought resistance by influencing the autophagy pathway. An Arabidopsis thaliana cost1 mutant has decreased growth and increased drought tolerance, together with constitutive autophagy and increased expression of drought-response genes, while overexpression of COST1 confers drought hypersensitivity and reduced autophagy. The COST1 protein is degraded upon plant dehydration, and this degradation is reduced upon treatment with inhibitors of the 26S proteasome or autophagy pathways. The drought resistance of a cost1 mutant is dependent on an active autophagy pathway, but independent of other known drought signaling pathways, indicating that COST1 acts through regulation of autophagy. In addition, COST1 colocalizes to autophagosomes with the autophagosome marker ATG8e and the autophagy adaptor NBR1, and affects the level of ATG8e protein through physical interaction with ATG8e, indicating a pivotal role in direct regulation of autophagy. We propose a model in which COST1 represses autophagy under optimal conditions, thus allowing plant growth. Under drought, COST1 is degraded, enabling activation of autophagy and suppression of growth to enhance drought tolerance. Our research places COST1 as an important regulator controlling the balance between growth and stress responses via the direct regulation of autophagy.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Estresse Fisiológico/fisiologia , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Autofagossomos/metabolismo , Autofagia/fisiologia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas de Transporte/metabolismo , Secas , Genes de Plantas , Transdução de Sinais , Estresse Fisiológico/genéticaRESUMO
The use of indium in semiconductor products has increased markedly in recent years. The release of indium into the ecosystem is inevitable. Under such circumstances, effective and accurate assessment of indium risk is important. An indispensable aspect of indium risk assessment is to understand the interactions of indium with plants, which are fundamental components of all ecosystems. Physiological responses of Arabidopsis thaliana exposed to indium were investigated by monitoring toxic effects, accumulation and speciation of indium in the plant. Indium can be taken up by plants and is accumulated mainly in roots. Limited indium root-to-shoot translocation occurs because of immobilization of indium in the root intercellular space and blockage of indium by the Casparian band in the endodermis. Indium caused stunted growth, oxidative stress, anthocyanization and unbalanced phosphorus nutrition. Indium jeopardizes phosphate uptake and translocation by inhibiting the accumulation of phosphate transporters PHOSPHATE TRANSPORTER1 (PHT1;1/4), responsible for phosphate uptake, and PHOSPHATE1 (PHO1), responsible for phosphate xylem loading. Organic acid secretion is stimulated by indium exposure. Secreted citrate could function as a potential detoxifier to lower indium uptake. Our findings provide insights into the potential fate and effects of indium in plants and will aid the evaluation of risks with indium contamination.
Assuntos
Arabidopsis/efeitos dos fármacos , Índio/toxicidade , Proteínas de Arabidopsis/metabolismo , Ácido Cítrico/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/metabolismo , Testes de ToxicidadeRESUMO
Calmodulin (CaM) is an important intracellular protein that binds Ca2+ and functions as a critical second messenger involved in numerous biological activities through extensive interactions with proteins and peptides. CaM's ability to adapt to binding targets with different structures is related to the flexible central helix separating the N- and C-terminal lobes, which allows for conformational changes between extended and collapsed forms of the protein. CaM-binding targets are most often identified using prediction algorithms that utilize sequence and structural data to predict regions of peptides and proteins that can interact with CaM. In this review, we provide an overview of different CaM-binding proteins, the motifs through which they interact with CaM, and shared properties that make them good binding partners for CaM. Additionally, we discuss the historical and current methods for predicting CaM binding, and the similarities and differences between these methods and their relative success at prediction. As new CaM-binding proteins are identified and classified, we will gain a broader understanding of the biological processes regulated through changes in Ca2+ concentration through interactions with CaM.
Assuntos
Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Cálcio/química , Calmodulina/química , Análise por Conglomerados , Análise Discriminante , Humanos , Aprendizado de Máquina , Cadeias de Markov , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Máquina de Vetores de SuporteRESUMO
As a most abundant plasticizer, Di-(2-ethylhexyl) phthalate (DEHP) has been widely used in agriculture with an associated potential toxicity to many species including plants via the production of the excessive reactive oxygen species (ROS). However, the potential toxic mechanisms of the plasticizer DEHP-induced oxidative damage to plants remain unknown. The antioxidant enzyme glutathione peroxidase has been suggested as biomarkers to reflect over excessive oxidative stress. In this study, the effect of DEHP on AtGPX6 was evaluated by multi-spectroscopic techniques and molecular docking method. The fluorescence intensity of AtGPX6 was reduced by the static quenching mechanism upon the addition of DEHP. The predominant forces in complex formation was mainly impelled by hydrogen bonding and Van der Waals forces based on the negative ΔH and ΔS, which was in accordance with the molecular docking results. In addition, the secondary structural changes resulted from the complex formation were investigated in presence of different amounts of DEHP by the combination of fluorescence, UV-vis absorption and Circular dichroism spectra, which revealed the loosening and unfolding of the framework of AtGPX6 accompanied with the enhancement of the hydrophilicity around the tryptophan residues. The exploration of the interaction mechanism of DEHP with AtGPX6 at molecular level would help to evaluate the toxicity of the plasticizers and forecast the related adverse effects on plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Biomarcadores/metabolismo , Dietilexilftalato/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Plastificantes/toxicidade , Medição de Risco/métodos , Antioxidantes/metabolismo , Arabidopsis/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Biosafety is a multidisciplinary approach that encompasses social, societal, ethical issues and policies for the regulations of genetically modified (GM) organisms. The potential health risks associated with GM sugarcane containing AVP1 gene confers resistance against drought and salinity were evaluated by animal feeding studies and some genotoxicity assays. Acute and sub-chronic toxicity examinations were carried out via oral dose administration of GM sugarcane juice supplemented with the normal diet (modified from certified rodent standard diet) on Wistar rats. AVP1 protein concentration in sugarcane juice was 1mg/1â¯mL. Biochemical, haematological blood analyses were performed and the results revealed that there were non-significant differences among all the treatment groups; GM sugarcane juice, non-GM sugarcane juice and the control group (normal diet and water). Genotoxicity assessment based on the comet assay and the micronucleus assay data exhibited that AVP1 GM sugarcane was not genotoxic or cytotoxic in rat's peripheral blood. These research findings supported the conclusion that GM AVP1 sugarcane was non-toxic in experimental animals. Therefore, data generated through this research work would be helpful for the commercial release of GM AVP1 sugarcane.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pirofosfatase Inorgânica/genética , Pirofosfatase Inorgânica/metabolismo , Saccharum/genética , Animais , Proteínas de Arabidopsis/toxicidade , Peso Corporal , Ensaio Cometa , Feminino , Regulação da Expressão Gênica de Plantas , Pirofosfatase Inorgânica/toxicidade , Masculino , Valor Nutritivo , Plantas Geneticamente Modificadas , Ratos , Ratos Wistar , Testes de ToxicidadeRESUMO
The identification of plant-pathogen protein-protein interactions (PPIs) is an attractive and challenging research topic for deciphering the complex molecular mechanism of plant immunity and pathogen infection. Considering that the experimental identification of plant-pathogen PPIs is time-consuming and labor-intensive, computational methods are emerging as an important strategy to complement the experimental methods. In this work, we first evaluated the performance of traditional computational methods such as interolog, domain-domain interaction and domain-motif interaction in predicting known plant-pathogen PPIs. Owing to the low sensitivity of the traditional methods, we utilized Random Forest to build an inter-species PPI prediction model based on multiple sequence encodings and novel network attributes in the established plant PPI network. Critical assessment of the features demonstrated that the integration of sequence information and network attributes resulted in significant and robust performance improvement. Additionally, we also discussed the influence of Gene Ontology and gene expression information on the prediction performance. The Web server implementing the integrated prediction method, named InterSPPI, has been made freely available at http://systbio.cau.edu.cn/intersppi/index.php. InterSPPI could achieve a reasonably high accuracy with a precision of 73.8% and a recall of 76.6% in the independent test. To examine the applicability of InterSPPI, we also conducted cross-species and proteome-wide plant-pathogen PPI prediction tests. Taken together, we hope this work can provide a comprehensive understanding of the current status of plant-pathogen PPI predictions, and the proposed InterSPPI can become a useful tool to accelerate the exploration of plant-pathogen interactions.
Assuntos
Proteínas de Plantas/metabolismo , Plantas/metabolismo , Plantas/microbiologia , Mapeamento de Interação de Proteínas/métodos , Algoritmos , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Biologia Computacional/métodos , Bases de Dados de Proteínas/estatística & dados numéricos , Perfilação da Expressão Gênica/estatística & dados numéricos , Ontologia Genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Aprendizado de Máquina , Modelos Biológicos , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Plantas/genética , Mapeamento de Interação de Proteínas/estatística & dados numéricosRESUMO
A major challenge in the field of proteomics is obtaining high-quality peptides for comprehensive proteome profiling by LC-MS. Here, evaluation and modification of a range of sample preparation methods using photosynthetically active Arabidopsis leaf tissue are done. It was found that inclusion of filter-aided sample preparation (FASP) based on filter digestion improves all protein extraction methods tested. Ultimately, a detergent-free urea-FASP approach that enables deep and robust quantification of leaf and root proteomes is shown. For example, from 4-day-old leaf tissue, up to 11 690 proteins were profiled from a single sample replicate. This method should be broadly applicable to researchers working with difficult to process plant samples.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Proteoma/metabolismo , Manejo de Espécimes/métodos , Arabidopsis/crescimento & desenvolvimento , Detergentes/química , Filtração , Folhas de Planta/crescimento & desenvolvimento , Raízes de Plantas/crescimento & desenvolvimento , Proteoma/análiseRESUMO
Plants use a tightly regulated immune system to fight off various pathogens. Phospholipase D (PLD) and its product, phosphatidic acid, have been shown to influence plant immunity; however, the underlying mechanisms remain unclear. Here, we show that the Arabidopsis mutants pldα1 and pldδ, respectively, exhibited enhanced resistance and enhanced susceptibility to both well-adapted and poorly adapted powdery mildew pathogens, and a virulent oomycete pathogen, indicating that PLDα1 negatively while PLDδ positively modulates post-penetration resistance. The pldα1δ double mutant showed a similar infection phenotype to pldα1, genetically placing PLDα1 downstream of PLDδ. Detailed genetic analyses of pldδ with mutations in genes for salicylic acid (SA) synthesis (SID2) and/or signaling (EDS1 and PAD4), measurement of SA and jasmonic acid (JA) levels, and expression of their respective reporter genes indicate that PLDδ contributes to basal resistance independent of EDS1/PAD4, SA, and JAsignaling. Interestingly, while PLDα1-enhanced green fluorescent protein (eGFP) was mainly found in the tonoplast before and after haustorium invasion, PLDδ-eGFP's focal accumulation to the plasma membrane around the fungal penetration site appeared to be suppressed by adapted powdery mildew. Together, our results demonstrate that PLDα1 and PLDδ oppositely modulate basal, post-penetration resistance against powdery mildew through a non-canonical mechanism that is independent of EDS1/PAD4, SA, and JA.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Ascomicetos/fisiologia , Fosfolipase D/metabolismo , Doenças das Plantas/imunologia , Ácido Salicílico/metabolismo , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Fosfolipase D/economia , Fosfolipase D/genética , Doenças das Plantas/microbiologia , Imunidade VegetalRESUMO
ß-Aminobutyric acid (BABA) induces broad-spectrum disease resistance, but also represses plant growth, which has limited its exploitation in crop protection. BABA perception relies on binding to the aspartyl-tRNA synthetase (AspRS) IBI1, which primes the enzyme for secondary defense activity. This study aimed to identify structural BABA analogues that induce resistance without stunting plant growth. Using site-directed mutagenesis, we demonstrate that the (l)-aspartic acid-binding domain of IBI1 is critical for BABA perception. Based on interaction models of this domain, we screened a small library of structural BABA analogues for growth repression and induced resistance against biotrophic Hyaloperonospora arabidopsidis (Hpa). A range of resistance-inducing compounds were identified, of which (R)-ß-homoserine (RBH) was the most effective. Surprisingly, RBH acted through different pathways than BABA. RBH-induced resistance (RBH-IR) against Hpa functioned independently of salicylic acid, partially relied on camalexin, and was associated with augmented cell wall defense. RBH-IR against necrotrophic Plectosphaerella cucumerina acted via priming of ethylene and jasmonic acid defenses. RBH-IR was also effective in tomato against Botrytis cinerea. Metabolic profiling revealed that RBH, unlike BABA, does not majorly affect plant metabolism. RBH primes distinct defense pathways against biotrophic and necrotrophic pathogens without stunting plant growth, signifying strong potential for exploitation in crop protection.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/imunologia , Desenvolvimento Vegetal , Imunidade Vegetal , Aminobutiratos/farmacologia , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Simulação por Computador , Resistência à Doença/efeitos dos fármacos , Etilenos/metabolismo , Fungos/fisiologia , Homosserina/farmacologia , Indóis/metabolismo , Solanum lycopersicum/efeitos dos fármacos , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/microbiologia , Mutação/genética , Desenvolvimento Vegetal/efeitos dos fármacos , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/efeitos dos fármacos , Domínios Proteicos , Ácido Salicílico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiazóis/metabolismoRESUMO
Iron (Fe) is an essential element for plants, utilized in nearly every cellular process. Because the adjustment of uptake under Fe limitation cannot satisfy all demands, plants need to acclimate their physiology and biochemistry, especially in their chloroplasts, which have a high demand for Fe. To investigate if a program exists for the utilization of Fe under deficiency, we analyzed how hydroponically grown Arabidopsis (Arabidopsis thaliana) adjusts its physiology and Fe protein composition in vegetative photosynthetic tissue during Fe deficiency. Fe deficiency first affected photosynthetic electron transport with concomitant reductions in carbon assimilation and biomass production when effects on respiration were not yet significant. Photosynthetic electron transport function and protein levels of Fe-dependent enzymes were fully recovered upon Fe resupply, indicating that the Fe depletion stress did not cause irreversible secondary damage. At the protein level, ferredoxin, the cytochrome-b6f complex, and Fe-containing enzymes of the plastid sulfur assimilation pathway were major targets of Fe deficiency, whereas other Fe-dependent functions were relatively less affected. In coordination, SufA and SufB, two proteins of the plastid Fe-sulfur cofactor assembly pathway, were also diminished early by Fe depletion. Iron depletion reduced mRNA levels for the majority of the affected proteins, indicating that loss of enzyme was not just due to lack of Fe cofactors. SufB and ferredoxin were early targets of transcript down-regulation. The data reveal a hierarchy for Fe utilization in photosynthetic tissue and indicate that a program is in place to acclimate to impending Fe deficiency.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Deficiências de Ferro , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , Cloroplastos/efeitos da radiação , Transporte de Elétrons/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Ferro/metabolismo , Luz , Fotossíntese/efeitos da radiação , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
A dynamic model of leaf CO2 assimilation was developed as an extension of the canonical steady-state model, by adding the effects of energy-dependent non-photochemical quenching (qE), chloroplast movement, photoinhibition, regulation of enzyme activity in the Calvin cycle, metabolite concentrations, and dynamic CO2 diffusion. The model was calibrated and tested successfully using published measurements of gas exchange and chlorophyll fluorescence on Arabidopsis thaliana ecotype Col-0 and several photosynthetic mutants and transformants affecting the regulation of Rubisco activity (rca-2 and rwt43), non-photochemical quenching (npq4-1 and npq1-2), and sucrose synthesis (spsa1). The potential improvements on CO2 assimilation under fluctuating irradiance that can be achieved by removing the kinetic limitations on the regulation of enzyme activities, electron transport, and stomatal conductance were calculated in silico for different scenarios. The model predicted that the rates of activation of enzymes in the Calvin cycle and stomatal opening were the most limiting (up to 17% improvement) and that effects varied with the frequency of fluctuations. On the other hand, relaxation of qE and chloroplast movement had a strong effect on average low-irradiance CO2 assimilation (up to 10% improvement). Strong synergies among processes were found, such that removing all kinetic limitations simultaneously resulted in improvements of up to 32%.
Assuntos
Arabidopsis/metabolismo , Dióxido de Carbono/metabolismo , Modelos Biológicos , Folhas de Planta/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Calibragem , Clorofila/metabolismo , Transporte de Elétrons , Luz , Complexos de Proteínas Captadores de Luz/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Mutação , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Estômatos de Plantas/fisiologia , Ribulose-Bifosfato Carboxilase/economia , Ribulose-Bifosfato Carboxilase/metabolismoRESUMO
Nitrogen (N) is a major macronutrient that is essential for plant growth. It is important for us to understand the key genes that are involved in the regulation of N utilization. In this study, we focused on a GARP-type transcription factor known as NSR1/MYR2, which has been reported to be induced under N-deficient conditions. Our results demonstrated that NSR1/MYR2 has a transcriptional repression activity and is specifically expressed in vascular tissues, especially in phloem throughout the plant under daily light-dark cycle regulation. The overexpression of NSR1/MYR2 delays nutrient starvation- and dark-triggered senescence in the mature leaves of excised whole aerial parts of Arabidopsis plants. Furthermore, the expression of asparagine synthetase 1 (ASN1), which plays an important role in N remobilization and reallocation, i.e. N reutilization, in Arabidopsis, is negatively regulated by NSR1/MYR2, since the expressions of NSR1/MYR2 and ASN1 were reciprocally regulated during the light-dark cycle and ASN1 expression was down-regulated in overexpressors of NSR1/MYR2 and up-regulated in T-DNA insertion mutants of NSR1/MYR2. Therefore, the present results suggest that NSR1/MYR2 plays a role in N reutilization as a negative regulator through controlling ASN1 expression.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Aspartato-Amônia Ligase/metabolismo , Nitrogênio/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/economia , Proteínas de Arabidopsis/genética , Aspartato-Amônia Ligase/genética , Transporte Biológico , Floema/metabolismo , Fotoperíodo , Folhas de Planta/metabolismo , Fatores de Transcrição/genéticaRESUMO
A gene regulatory network links transcription factors to their target genes and represents a map of transcriptional regulation. Much progress has been made in deciphering gene regulatory networks computationally. However, gene regulatory network inference for most eukaryotic organisms remain challenging. To improve the accuracy of gene regulatory network inference and facilitate candidate selection for experimentation, we developed an algorithm called GRACE (Gene Regulatory network inference ACcuracy Enhancement). GRACE exploits biological a priori and heterogeneous data integration to generate high- confidence network predictions for eukaryotic organisms using Markov Random Fields in a semi-supervised fashion. GRACE uses a novel optimization scheme to integrate regulatory evidence and biological relevance. It is particularly suited for model learning with sparse regulatory gold standard data. We show GRACE's potential to produce high confidence regulatory networks compared to state of the art approaches using Drosophila melanogaster and Arabidopsis thaliana data. In an A. thaliana developmental gene regulatory network, GRACE recovers cell cycle related regulatory mechanisms and further hypothesizes several novel regulatory links, including a putative control mechanism of vascular structure formation due to modifications in cell proliferation.