Shedding Light on the Dynamic Role of the "Target of Rapamycin" Kinase in the Fast-Growing C4 Species Setaria viridis, a Suitable Model for Biomass Crops.

The Target of Rapamycin (TOR) kinase pathway integrates energy and nutrient availability into metabolism promoting growth in eukaryotes. The overall higher efficiency on nutrient use translated into faster growth rates in C4 grass plants led to the investigation of differential transcriptional and metabolic responses to short-term chemical TOR complex (TORC) suppression in the model Setaria viridis. In addition to previously described responses to TORC inhibition (i.e., general growth arrest, translational repression, and primary metabolism reprogramming) in Arabidopsis thaliana (C3), the magnitude of changes was smaller in S. viridis, particularly regarding nutrient use efficiency and C allocation and partitioning that promote biosynthetic growth. Besides photosynthetic differences, S. viridis and A. thaliana present several specificities that classify them into distinct lineages, which also contribute to the observed alterations mediated by TOR. Indeed, cell wall metabolism seems to be distinctly regulated according to each cell wall type, as synthesis of non-pectic polysaccharides were affected in S. viridis, whilst assembly and structure in A. thaliana. Our results indicate that the metabolic network needed to achieve faster growth seems to be less stringently controlled by TORC in S. viridis.

The Contribution of Metabolomics to Systems Biology: Current Applications Bridging Genotype and Phenotype in Plant Science.

Metabolomics is a valuable approach used to acquire comprehensive information about the set of metabolites in a cell or tissue, enabling a functional screen of the cellular activities in biological systems. Although metabolomics provides a more immediate and dynamic picture of phenotypes in comparison to the other omics, it is also the most complicated to measure because no single analytical technology can capture the extraordinary complexity of metabolite diversity in terms of structure and physical properties. Metabolomics has been extensively employed for a wide range of applications in plant science, which will be described in detail in this chapter. Among them, metabolomics is used for discriminating patterns of plant responses to genetic and environmental perturbations, as diagnostics and prediction tool to elucidate the function of genes for important and complex agronomic traits in crop species, and flux measurements are used to dissect the structure and regulatory properties of metabolic networks.

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions.

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

The Angiotensin Converting Enzyme 2 (ACE2), Gut Microbiota, and Cardiovascular Health.

BACKGROUND: The renin-angiotensin system (RAS) is an important enzymatic system responsible for the regulation of biological functions, such as the arterial pressure, hydroelectrolytic control, vascular vasodilatation/vasoconstriction and more recently metabolic functions. OBJECTIVES: The aim of the present review is to discuss the associations between the gut microbiome and the renin-angiotensin system and the influence of their intimate relationship on the cardiovascular health. METHODS: A literature review of the main studies published regarding the relationship among the renin-angiotensin system, gut microbiota and cardiovascular health was performed. RESULTS: The association between the ACE2 and gut microbiota has been discussed. It is shown that the ACE2/Ang 1-7 axis modulates the immune response, influencing the microbiota composition, and thus being one of the causes for some diseases physiophatologies, such as diarrhea and intestinal inflammatory disease. CONCLUSION: The association between RAS and gut microbiota seems to have a strong influence on the genesis of cardiovascular diseases, through direct mechanisms, such as nerve stimulation, or indirectly on metabolic parameters, such as weight, adiposity and lipid profile.

Selection of reliable reference genes for RT-qPCR analysis during developmental stages and abiotic stress in Setaria viridis.

Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but reliable results depend on the use of stable reference genes for proper normalization. This study proposed to test the expression stability of 13 candidate reference genes in Setaria viridis, a monocot species recently proposed as a new C4 model plant. Gene expression stability of these genes was assayed across different tissues and developmental stages of Setaria and under drought or aluminum stress. In general, our results showed Protein Kinase, RNA Binding Protein and SDH as the most stable genes. Moreover, pairwise analysis showed that two reference genes were sufficient to normalize the gene expression data under each condition. By contrast, GAPDH and ACT were the least stably expressed genes tested. Validation of suitable reference genes was carried out to profile the expression of P5CS and GolS during abiotic stress. In addition, normalization of gene expression of SuSy, involved in sugar metabolism, was assayed in the developmental dataset. This study provides a list of reliable reference genes for transcript normalization in S. viridis in different tissues and stages of development and under abiotic stresses, which will facilitate genetic studies in this monocot model plant.

Candidatus Liberibacter americanus induces significant reprogramming of the transcriptome of the susceptible citrus genotype.

BACKGROUND: Citrus huanglongbing (HLB) disease is caused by endogenous, phloem-restricted, Gram negative, uncultured bacteria named Candidatus Liberibacter africanus (CaLaf), Ca. L. asiaticus (CaLas), and Ca. L. americanus (CaLam), depending on the continent where the bacteria were first detected. The Asian citrus psyllid vector, Diaphorina citri, transmits CaLas and CaLam and both Liberibacter species are present in Brazil. Several studies of the transcriptional response of citrus plants manifesting HLB symptoms have been reported, but only for CaLas infection. This study evaluated the transcriptional reprogramming of a susceptible genotype of sweet orange challenged with CaLam, using a customized 385K microarray containing approximately 32,000 unigene transcripts. We analyzed global changes in gene expression of CaLam-infected leaves of sweet orange during the symptomatic stage of infection and compared the results with previously published microarray studies that used CaLas-infected plants. Twenty candidate genes were selected to validate the expression profiles in symptomatic and asymptomatic PCR-positive leaves infected with CaLas or CaLam. RESULTS: The microarray analysis identified 633 differentially expressed genes during the symptomatic stage of CaLam infection. Among them, 418 (66%) were upregulated and 215 (34%) were down regulated. Five hundred and fourteen genes (81%) were orthologs of genes from Arabidopsis thaliana. Gene set enrichment analysis (GSEA) revealed that several of the transcripts encoded transporters associated with the endomembrane system, especially zinc transport. Among the most biologically relevant gene transcripts in GSEA were those related to signaling, metabolism and/or stimulus to hormones, genes responding to stress and pathogenesis, biosynthesis of secondary metabolites, oxidative stress and transcription factors belonging to different families. Real time PCR of 20 candidate genes validated the expression pattern of some genes in symptomatic and asymptomatic leaves infected with CaLam or CaLas. CONCLUSIONS: Many gene transcripts and biological processes are significantly altered upon CaLam infection. Some of them had been identified in response to CaLas infection, while others had not been previously reported. These data will be useful for selecting target genes for genetic engineering to control HLB.

Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.

Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress.

Global gene expression of Poncirus trifoliata, Citrus sunki and their hybrids under infection of Phytophthora parasitica.

BACKGROUND: Gummosis and root rot caused by Phytophthora are among the most economically important diseases in citrus. Four F1 resistant hybrids (Pool R), and four F1 susceptible hybrids (Pool S) to P. parasitica, were selected from a cross between susceptible Citrus sunki and resistant Poncirus trifoliata cv. Rubidoux. We investigated gene expression in pools of four resistant and four susceptible hybrids in comparison with their parents 48 hours after P. parasitica inoculation. We proposed that genes differentially expressed between resistant and susceptible parents and between their resistant and susceptible hybrids provide promising candidates for identifying transcripts involved in disease resistance. A microarray containing 62,876 UniGene transcripts selected from the CitEST database and prepared by NimbleGen Systems was used for analyzing global gene expression 48 hours after infection with P. parasitica. RESULTS: Three pairs of data comparisons (P. trifoliata/C. sunki, Pool R/C. sunki and Pool R/Pool S) were performed. With a filter of false-discovery rate less than 0.05 and fold change greater than 3.0, 21 UniGene transcripts common to the three pairwise comparative were found to be up-regulated, and 3 UniGene transcripts were down-regulated. Among them, our results indicated that the selected transcripts were probably involved in the whole process of plant defense responses to pathogen attack, including transcriptional regulation, signaling, activation of defense genes participating in HR, single dominant genes (R gene) such as TIR-NBS-LRR and RPS4 and switch of defense-related metabolism pathway. Differentially expressed genes were validated by RT-qPCR in susceptible and resistant plants and between inoculated and uninoculated control plants CONCLUSIONS: Twenty four UniGene transcripts were identified as candidate genes for Citrus response to P. parasitica. UniGene transcripts were likely to be involved in disease resistance, such as genes potentially involved in secondary metabolite synthesis, intracellular osmotic adjustment, signal transduction pathways of cell death, oxidative burst and defense gene expression. Furthermore, our microarray data suggest another type of resistance in Citrus-Phytophthora interaction conferred by single dominant genes (R gene) since we encountered two previously reported R genes (TIR-NBS-LRR and RPS4) upregulated in the resistant genotypes relative to susceptible. We identified 7 transcripts with homology in other plants but yet unclear functional characterization which are an interesting pool for further analyses and 3 transcripts where no significant similarity was found. This is the first microarray study addressing an evaluation of transcriptional changes in response to P. parasitica in Citrus.