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Functional annotation based on Gene Ontology has provided a structured and comprehensive system to access the current knowledge about the function of genes. For model plants such as Arabidopsis thaliana, there is a constant updating and restructuring of the functional annotation that increases the reliability of the analyses that use it. For tomato (Solanum lycopersicum), a crop widely used as a model plant for the study of fleshy fruits, there is no functional annotation, at least not freely accessible, even though its genome has long been sequenced and annotated. In this work, we generated, using a simplified version of the maize GAMER pipeline, a tomato Gene Ontology functional annotation with 72.42% (ITAG3.2) and 74.2% (ITAG4.0) of protein-coding genes with at least one GO term association. With this dataset, we share a reliable and easy-to-use tool with the tomato community.
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Huanglongbing (HLB) is one of the most destructive diseases threatening citriculture worldwide. This disease has been associated with α-proteobacteria species, namely Candidatus Liberibacter. Due to the unculturable nature of the causal agent, it has been difficult to mitigate the disease, and nowadays a cure is not available. MicroRNAs (miRNAs) are key regulators of gene expression, playing an essential role in abiotic and biotic stress in plants including antibacterial responses. However, knowledge derived from non-model systems including Candidatus Liberibacter asiaticus (CLas)-citrus pathosystem remains largely unknown. In this study, small RNA profiles from Mexican lime (Citrus aurantifolia) plants infected with CLas at asymptomatic and symptomatic stages were generated by sRNA-Seq, and miRNAs were obtained with ShortStack software. A total of 46 miRNAs, including 29 known miRNAs and 17 novel miRNAs, were identified in Mexican lime. Among them, six miRNAs were deregulated in the asymptomatic stage, highlighting the up regulation of two new miRNAs. Meanwhile, eight miRNAs were differentially expressed in the symptomatic stage of the disease. The target genes of miRNAs were related to protein modification, transcription factors, and enzyme-coding genes. Our results provide new insights into miRNA-mediated regulation in C. aurantifolia in response to CLas infection. This information will be useful to understand molecular mechanisms behind the defense and pathogenesis of HLB.
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Desiccation tolerance is an ancient and complex trait that spans all major lineages of life on earth. Although important in the evolution of land plants, the mechanisms that underlay this complex trait are poorly understood, especially for vegetative desiccation tolerance (VDT). The lack of suitable closely related plant models that offer a direct contrast between desiccation tolerance and sensitivity has hampered progress. We have assembled high-quality genomes for two closely related grasses, the desiccation-tolerant Sporobolus stapfianus and the desiccation-sensitive Sporobolus pyramidalis Both species are complex polyploids; S. stapfianus is primarily tetraploid, and S. pyramidalis is primarily hexaploid. S. pyramidalis undergoes a major transcriptome remodeling event during initial exposure to dehydration, while S. stapfianus has a muted early response, with peak remodeling during the transition between 1.5 and 1.0 grams of water (gH2O) g-1 dry weight (dw). Functionally, the dehydration transcriptome of S. stapfianus is unrelated to that for S. pyramidalis A comparative analysis of the transcriptomes of the hydrated controls for each species indicated that S. stapfianus is transcriptionally primed for desiccation. Cross-species comparative analyses indicated that VDT likely evolved from reprogramming of desiccation tolerance mechanisms that evolved in seeds and that the tolerance mechanism of S. stapfianus represents a recent evolution for VDT within the Chloridoideae. Orthogroup analyses of the significantly differentially abundant transcripts reconfirmed our present understanding of the response to dehydration, including the lack of an induction of senescence in resurrection angiosperms. The data also suggest that failure to maintain protein structure during dehydration is likely critical in rendering a plant desiccation sensitive.
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Adaptação Fisiológica/genética , Poaceae/genética , Dessecação/métodos , Genômica/métodos , Folhas de Planta/genética , Proteínas de Plantas/genética , Água/metabolismoRESUMO
Transcription factors are important regulators of gene expression. They can orchestrate the activation or repression of hundreds or thousands of genes and control diverse processes in a coordinated way. This work explores the effect of a master regulator of plant development, BOLITA (BOL), in plant metabolism, with a special focus on specialized metabolism. For this, we used an Arabidopsis thaliana line in which the transcription factor activity can be induced. Fingerprinting metabolomic analyses of whole plantlets were performed at different times after induction. After 96 h, all induced replicas clustered as a single group, in contrast with all controls which did not cluster. Metabolomic analyses of shoot and root tissues enabled the putative identification of differentially accumulated metabolites in each tissue. Finally, the analysis of global gene expression in induced vs. non-induced root samples, together with enrichment analyses, allowed the identification of enriched metabolic pathways among the differentially expressed genes and accumulated metabolites after the induction. We concluded that the induction of BOL activity can modify the Arabidopsis metabolome. Future work should investigate whether its action is direct or indirect, and the implications of the metabolic changes for development regulation and bioprospection.
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Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Metaboloma , Fatores de Transcrição/metabolismo , Arabidopsis , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/genética , TranscriptomaRESUMO
BACKGROUND: Nitrogen (N) and phosphorus (P) are macronutrients essential for crop growth and productivity. In cultivated fields, N and P levels are rarely sufficient, contributing to the gap between realized and potential production. Fertilizer application increases nutrient availability, but is not available to all farmers, nor are current rates of application sustainable or environmentally desirable. Transcriptomic studies of cereal crops have revealed dramatic responses to either low N or low P single stress treatments. In the field, however, levels of both N and P may be suboptimal. The interaction between N and P starvation responses remains to be fully characterized. RESULTS: We characterized growth and root and leaf transcriptomes of young maize plants under nutrient replete, low N, low P or combined low NP conditions. We identified 1555 genes to respond to our nutrient treatments, in one or both tissues. A large group of genes, including many classical P starvation response genes, were regulated antagonistically between low N and P conditions. An additional experiment over a range of N availability indicated that a mild reduction in N levels was sufficient to repress the low P induction of P starvation genes. Although expression of P transporter genes was repressed under low N or low NP, we confirmed earlier reports of P hyper accumulation under N limitation. CONCLUSIONS: Transcriptional responses to low N or P were distinct, with few genes responding in a similar way to the two single stress treatments. In combined NP stress, the low N response dominated, and the P starvation response was largely suppressed. A mild reduction in N availability was sufficient to repress the induction of P starvation associated genes. We conclude that activation of the transcriptional response to P starvation in maize is contingent on N availability.
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Nitrogênio/farmacologia , Fósforo/farmacologia , Zea mays/efeitos dos fármacos , Zea mays/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Nitrogênio/administração & dosagem , Fósforo/administração & dosagem , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plântula/crescimento & desenvolvimento , Estresse Fisiológico/efeitos dos fármacos , Zea mays/metabolismoRESUMO
BACKGROUND: Trichoderma species are among the most effective cell factories to produce recombinant proteins, whose productivity relies on the molecular toolkit and promoters available for the expression of the target protein. Although inducible promoter systems have been developed for producing recombinant proteins in Trichoderma, constitutive promoters are often a desirable alternative. Constitutive promoters are simple to use, do not require external stimuli or chemical inducers to be activated, and lead to purer enzyme preparations. Moreover, most of the promoters for homologous and heterologous expression reported in Trichoderma have been commonly evaluated by directly assessing production of industrial enzymes, requiring optimization of laborious protocols. RESULTS: Here we report the identification of Pccg6, a novel Trichoderma atroviride constitutive promoter, that has similar transcriptional strength as that of the commonly used pki1 promoter. Pccg6 displayed conserved arrangements of transcription factor binding sites between promoter sequences of Trichoderma ccg6 orthologues genes, potentially involved in their regulatory properties. The predicted ccg6-encoded protein potentially belongs to the SPE1/SPI1 protein family and shares high identity with CCG6 orthologue sequences from other fungal species including Trichoderma reesei, Trichoderma virens, Trichoderma asperellum, and to a lesser extent to that of Neurospora crassa. We also report the use of the Pccg6 promoter to drive the expression of PTXD, a phosphite oxidoreductase of bacterial origin, which allowed T. atroviride to utilize phosphite as a sole source of phosphorus. We propose ptxD as a growth reporter gene that allows real-time comparison of the functionality of different promoters by monitoring growth of Trichoderma transgenic lines and enzymatic activity of PTXD. Finally, we show that constitutive expression of ptxD provided T. atroviride a competitive advantage to outgrow bacterial contaminants when supplied with phosphite as a sole source of phosphorus. CONCLUSIONS: A new constitutive promoter, ccg6, for expression of homologous and heterologous proteins has been identified and tested in T. atroviride to express PTXD, which resulted in an effective and visible phenotype to evaluate transcriptional activity of sequence promoters. Use of PTXD as a growth marker holds great potential for assessing activity of other promoters and for biotechnological applications as a contamination control system.
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Genes Fúngicos , Regiões Promotoras Genéticas , Trichoderma/genética , Proteínas de Bactérias/genética , Clonagem Molecular , Oxirredutases/genética , Proteínas Recombinantes/genéticaRESUMO
Bioinformatic analysis of small RNA sequencing libraries consists of transforming a series of small RNA sequencing experiment fastq files into a table containing small RNA sequences and their abundance. This is achieved by cleaning the reads, aligning the cleaned reads to a reference, and parsing the alignment results. In this protocol we present the most common option, and the rationale, for each of these steps.
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MicroRNAs/genética , Algoritmos , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos , SoftwareRESUMO
After linear sequences of genomes and epigenomic landscape data, the 3D organization of chromatin in the nucleus is the next level to be explored. Different organisms present a general hierarchical organization, with chromosome territories at the top. Chromatin interaction maps, obtained by chromosome conformation capture (3C)-based methodologies, for eight plant species reveal commonalities, but also differences, among them and with animals. The smallest structures, found in high-resolution maps of the Arabidopsis genome, are single genes. Epigenetic marks (histone modification and DNA methylation), transcriptional activity, and chromatin interaction appear to be correlated, and whether structure is the cause or consequence of the function of interacting regions is being actively investigated.
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Cromossomos de Plantas/genética , Genoma de Planta/genética , Cromatina/genética , Epigênese Genética/genéticaRESUMO
BACKGROUND: Gene regulatory variation has been proposed to play an important role in the adaptation of plants to environmental stress. In the central highlands of Mexico, farmer selection has generated a unique group of maize landraces adapted to the challenges of the highland niche. In this study, gene expression in Mexican highland maize and a reference maize breeding line were compared to identify evidence of regulatory variation in stress-related genes. It was hypothesised that local adaptation in Mexican highland maize would be associated with a transcriptional signature observable even under benign conditions. METHODS: Allele specific expression analysis was performed using the seedling-leaf transcriptome of an F1 individual generated from the cross between the highland adapted Mexican landrace Palomero Toluqueño and the reference line B73, grown under benign conditions. Results were compared with a published dataset describing the transcriptional response of B73 seedlings to cold, heat, salt and UV treatments. RESULTS: A total of 2,386 genes were identified to show allele specific expression. Of these, 277 showed an expression difference between Palomero Toluqueño and B73 alleles under benign conditions that anticipated the response of B73 cold, heat, salt and/or UV treatments, and, as such, were considered to display a prior stress response. Prior stress response candidates included genes associated with plant hormone signaling and a number of transcription factors. Construction of a gene co-expression network revealed further signaling and stress-related genes to be among the potential targets of the transcription factors candidates. DISCUSSION: Prior activation of responses may represent the best strategy when stresses are severe but predictable. Expression differences observed here between Palomero Toluqueño and B73 alleles indicate the presence of cis-acting regulatory variation linked to stress-related genes in Palomero Toluqueño. Considered alongside gene annotation and population data, allele specific expression analysis of plants grown under benign conditions provides an attractive strategy to identify functional variation potentially linked to local adaptation.
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Plant interactions with arbuscular mycorrhizal fungi have long attracted interest for their potential to promote more efficient use of mineral resources in agriculture. Their use, however, remains limited by a lack of understanding of the processes that determine the outcome of the symbiosis. In this study, the impact of host genotype on growth response to mycorrhizal inoculation was investigated in a panel of diverse maize lines. A panel of 30 maize lines was evaluated with and without inoculation with arbuscular mycorrhizal fungi. The line Oh43 was identified to show superior response and, along with five other reference lines, was characterized in greater detail in a split-compartment system, using 33 P to quantify mycorrhizal phosphorus uptake. Changes in relative growth indicated variation in host capacity to profit from the symbiosis. Shoot phosphate content, abundance of root-internal and -external fungal structures, mycorrhizal phosphorus uptake, and accumulation of transcripts encoding plant PHT1 family phosphate transporters varied among lines. Superior response in Oh43 is correlated with extensive development of root-external hyphae, accumulation of specific Pht1 transcripts and high phosphorus uptake by mycorrhizal plants. The data indicate that host genetic factors influence fungal growth strategy with an impact on plant performance.
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Hifas/metabolismo , Micorrizas/metabolismo , Proteínas de Transporte de Fosfato/genética , Fósforo/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/microbiologia , Zea mays/genética , Zea mays/microbiologia , Biomassa , Regulação da Expressão Gênica de Plantas , Proteínas de Transporte de Fosfato/metabolismo , Desenvolvimento Vegetal , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Quantitative real-time RT-PCR (qRT-PCR) has become one of the most widely used methods for accurate quantification of gene expression. Since there are no universal reference genes for normalization, the optimal strategy to normalize raw qRT-PCR data is to perform an initial comparison of a set of independent reference genes to assess the most stable ones in each biological model. Normalization of a qRT-PCR experiment helps to ensure that the results are both statistically significant and biologically meaningful. Tomato is the model of choice to study fleshy fruit development. The miniature tomato (Solanum lycopersicum L.) cultivar Micro-Tom (MT) is considered a model system for tomato genetics and functional genomics. A new genotype, containing the Rg1 allele, improves tomato in vitro regeneration. In this work, we evaluated the expression stability of four tomato reference genes, namely CAC, SAND, Expressed, and ACTIN2. We showed that the genes CAC and Exp are the best reference genes of the four we tested during fruit development in the MT-Rg1 genotype. Furthermore, we validated the reference genes by showing that the expression profiles of the transcription factors FRUITFULL1 and APETALA2c during fruit development are comparable to previous reports using other tomato cultivars.
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Desiccation tolerance (DT) is a remarkable process that allows seeds in the dry state to remain viable for long periods of time that in some instances exceed 1,000 y. It has been postulated that seed DT evolved by rewiring the regulatory and signaling networks that controlled vegetative DT, which itself emerged as a crucial adaptive trait of early land plants. Understanding the networks that regulate seed desiccation tolerance in model plant systems would provide the tools to understand an evolutionary process that played a crucial role in the diversification of flowering plants. In this work, we used an integrated approach that included genomics, bioinformatics, metabolomics, and molecular genetics to identify and validate molecular networks that control the acquisition of DT in Arabidopsis seeds. Two DT-specific transcriptional subnetworks were identified related to storage of reserve compounds and cellular protection mechanisms that act downstream of the embryo development master regulators LEAFY COTYLEDON 1 and 2, FUSCA 3, and ABSCICIC ACID INSENSITIVE 3. Among the transcription factors identified as major nodes in the DT regulatory subnetworks, PLATZ1, PLATZ2, and AGL67 were confirmed by knockout mutants and overexpression in a desiccation-intolerant mutant background to play an important role in seed DT. Additionally, we found that constitutive expression of PLATZ1 in WT plants confers partial DT in vegetative tissues.
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Adaptação Fisiológica/genética , Arabidopsis/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Sementes/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dessecação , Ontologia Genética , Genômica/métodos , Metabolômica/métodos , Mutação , Plantas Geneticamente Modificadas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVE: Due to the increased availability of effective treatments, patients with systemic lupus erythematosus (SLE) now have longer survival times, and factors involved in cumulative chronic damage in SLE need to be better understood. This study was undertaken to evaluate the relationship between smoking and cumulative chronic damage in SLE patients. METHODS: A cross-sectional study of SLE patients was performed to investigate the possible association between smoking exposure (ever [previous or current, active or secondhand smokers] or never) and cumulative chronic damage as measured by the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SDI). A systematic review of the literature was conducted by cross-searching Medline for the terms lupus and smoking. RESULTS: We enrolled 105 patients with SLE (96% female), with a mean ± SD age of 40.7 ± 11.4 years and a mean followup time of 8.98 years. Of the 105 patients, 74 had an SDI score of 1-10, and 31 had an SDI score of 0. The difference between smoking exposure and no smoking exposure was significant (P = 0.02 by chi-square test in contingency table analysis), and SLE patients who were never exposed to smoking had 0.78 times the risk of progressing toward a cumulative damage status (SDI score of > 0) (95% confidence interval 0.16-0.98) throughout the followup period compared to those who were ever exposed. In the systematic review of the literature, we found only a small number of articles that addressed some aspects of the relationship between smoking exposure and cumulative damage in SLE patients. CONCLUSION: Our findings indicate that smoking exposure is associated with cumulative chronic damage, as determined by the SDI score, in patients with SLE. Smoking exposure may have deleterious effects on lupus morbidity, and more detailed studies of this association are needed.
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Lúpus Eritematoso Sistêmico/fisiopatologia , Fumar/epidemiologia , Poluição por Fumaça de Tabaco/estatística & dados numéricos , Adulto , Estudos de Coortes , Estudos Transversais , Feminino , Humanos , Estudos Longitudinais , Lúpus Eritematoso Sistêmico/epidemiologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Índice de Gravidade de DoençaRESUMO
Purple acid phosphatases (PAPs) play an important role in plant phosphorus nutrition, both by liberating phosphorus from organic sources in the soil and by modulating distribution within the plant throughout growth and development. Furthermore, members of the PAP protein family have been implicated in a broader role in plant mineral homeostasis, stress responses and development. We have identified 33 candidate PAP encoding gene models in the maize (Zea mays ssp. mays var. B73) reference genome. The maize Pap family includes a clear single-copy ortholog of the Arabidopsis gene AtPAP26, shown previously to encode both major intracellular and secreted acid phosphatase activities. Certain groups of PAPs present in Arabidopsis, however, are absent in maize, while the maize family contains a number of expansions, including a distinct radiation not present in Arabidopsis. Analysis of RNA-sequencing based transcriptome data revealed accumulation of maize Pap transcripts in multiple plant tissues at multiple stages of development, and increased accumulation of specific transcripts under low phosphorus availability. These data suggest the maize PAP family as a whole to have broad significance throughout the plant life cycle, while highlighting potential functional specialization of individual family members.
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Synchronized communication between gametophytic and sporophytic tissue is crucial for successful reproduction, and hormones seem to have a prominent role in it. Here, we studied the role of the Arabidopsis (Arabidopsis thaliana) cytochrome P450 CYP78A9 enzyme during reproductive development. First, controlled pollination experiments indicate that CYP78A9 responds to fertilization. Second, while CYP78A9 overexpression can uncouple fruit development from fertilization, the cyp78a8 cyp78a9 loss-of-function mutant has reduced seed set due to outer ovule integument development arrest, leading to female sterility. Moreover, CYP78A9 has a specific expression pattern in inner integuments in early steps of ovule development as well as in the funiculus, embryo, and integuments of developing seeds. CYP78A9 overexpression did not change the response to the known hormones involved in flower development and fruit set, and it did not seem to have much effect on the major known hormonal pathways. Furthermore, according to previous predictions, perturbations in the flavonol biosynthesis pathway were detected in cyp78a9, cyp78a8 cyp78a9, and empty siliques (es1-D) mutants. However, it appeared that they do not cause the observed phenotypes. In summary, these results add new insights into the role of CYP78A9 in plant reproduction and present, to our knowledge, the first characterization of metabolite differences between mutants in this gene family.