Aluminum triggers broad changes in microRNA expression in rice roots.

MicroRNAs are small 21-nucleotide RNA molecules with regulatory roles in development and in response to stress. Expression of some plant miRNAs has been specifically associated with responses to abiotic stresses caused by cold, light, iron, and copper ions. In acid soils, aluminum solubility increases, thereby causing severe damage to plants. Although physiological aspects of aluminum toxicity in plants have been well characterized, the molecular mediators are not fully elucidated. There have been no reports about miRNA responses to aluminum stress. Modulation of miRNA expression may constitute a key element to explain the mechanisms implicated in aluminum toxicity and tolerance. We examined the expression of at least one miRNA member from each miRNA family in rice roots of Oryza sativa spp indica cv. Embrapa Taim and Oryza sativa spp japonica cv. Nipponbare under high concentrations of aluminum. Forty-six miRNA families were effectively detected by quantitative PCR. Among these, 13 were down-regulated and six were up-regulated in roots of the Nipponbare cultivar after 8 h of aluminum treatment. In roots of the Embrapa Taim cultivar, five miRNAs were down-regulated and three were up-regulated. Analyses of their putative targets suggest that these rice miRNAs are involved in the regulation of various metabolic pathways in response to high concentrations of aluminum.

Transcriptional profiling and physiological responses reveal new insights into drought tolerance in a semiarid adapted species, Anacardium occidentale.

Water stress affects plant performance at various organisational levels, from morphological to molecular, with a drastic drop in crop yield. Integrative studies involving transcriptomics and physiological data in recognized tolerant species are appropriate strategies to identify and understand molecular and functional processes related to water deficit tolerance. The cashew tree (Anacardium occidentale) is a species naturally adapted to environments with low water availability associated with adverse conditions such as heat, high radiation and salinity. We used an integrative strategy, combining classical physiological measurements with high throughput RNA-seq to understand the main adaptive mechanisms of cashew to water deficit followed by recovery. Physiological analyses indicate that young cashew plants display typical isohydric behaviour. They first exhibit rapid stomatal closure, followed by CO2 assimilation, thus preserving the relative water content, membrane integrity and photosystem II activity. Differential expression was observed in 1733 genes from plant leaves exposed to water deficit stress for 26 days. Among them, 705 were upregulated and 1028 were downregulated. After rewatering, 1330 (76.7%) genes returned to their basal expression level. Transcriptional, combined with physiological data, reveal that cashew plants display high phenotypic plasticity and resilience to acute water deficit, and do not activate senescence pathways. A series of genes/pathways and processes involved with drought tolerance in cashew are evidenced, particularly in carbon metabolism, photosynthesis and chloroplast homeostasis.

De novo assembly of Vriesea carinata leaf transcriptome to identify candidate cysteine-proteases.

Vriesea carinata is an endemic bromeliad from the Brazilian Atlantic Forest. It has trichome and tank system in their leaves which allows to absorb water and nutrients. It belongs to Bromeliaceae family, which includes several species highly enriched of cysteine-proteases (CysPs). These proteolytic enzymes regulate processes as senescence, cell differentiation, pathogen-linked programmed cell death and mobilization of proteins. Although, their biological importance, there are not genomic resources in V. carinata that can help to identify and understand their molecular mechanisms involved in different biological processes. Thus high-throughput transcriptome sequencing of V. carinata is necessary to generate sequences for the purpose of gene discovery and functional genomic studies. In the present study, we sequenced and assembled the V. carinata transcriptome to the identification of CysPs. A total of 43,232 contigs were assembled for the leaf tissue. BLAST analysis indicated that 23,803 contigs exhibited similarity to non-redundant Viridiplantae proteins. 28.24% of the contigs were classified into the COG database, and gene ontology categorized them into 61 functional groups. A metabolic pathway analysis with KEGG revealed 9679 contigs assigned to 31 metabolic pathways. Among 16 full-length CysPs identified, 11 were evaluated in respect to their expression patterns in the leaf apex, base and inflorescence tissues. The results showed differential expression levels of legumain, metacaspase, pyroglutamyl and papain-like CysPs depending of the leaf region. These results provide a global overview of V. carinata gene functions and expression activities of CysPs in those tissues.

Novel and conserved microRNAs in soybean floral whorls.

MicroRNAs (miRNAs) correspond to a class of endogenous small non-coding RNAs (19-24 nt) that regulates the gene expression, through mRNA target cleavage or translation inhibition. In plants, miRNAs have been shown to play pivotal roles in a wide variety of metabolic and biological processes like plant growth, development, and response to biotic and abiotic stress. Soybean is one of the most important crops worldwide, due to the production of oil and its high protein content. The reproductive phase is considered the most important for soybean yield, which is mainly intended to produce the grains. The identification of miRNAs is not yet saturated in soybean, and there are no studies linking them to the different floral organs. In this study, three different mature soybean floral whorls were used in the construction of sRNA libraries. The sequencing of petal, carpel and stamen libraries generated a total of 10,165,661 sequences. Subsequent analyses identified 200 miRNAs sequences, among which, 41 were novel miRNAs, 80 were conserved soybean miRNAs, 31 were new antisense conserved soybean miRNAs and 46 were soybean miRNAs isoforms. We also found a new miRNA conserved in other plant species, and finally one miRNA-sibling of a soybean conserved miRNA. Conserved and novel miRNAs were evaluated by RT-qPCR. We observed a differential expression across the three whorls for six miRNAs. Computational predicted targets for miRNAs analyzed by RT-qPCR were identified and present functions related to reproductive process in plants. In summary, the increased accumulation of specific and novel miRNAs in different whorls indicates that miRNAs are an important part of the regulatory network in soybean flower.

Retinoid-mediated induction of the fat-storing phenotype in a liver connective tissue cell line (GRX).

The GRX cell line is derived from murine liver connective tissue cells. It has myofibroblastic characteristics and can be induced to display a phenotype analogous to fat-storing (Ito) cells. Retinol-mediated induction of the fat-storing phenotype was studied in vitro. Based on the incorporation of radiolabelled acetate into cell lipids, cholesterol synthesis increased and phospholipid synthesis was modified shortly after the beginning of the induction, indicating an activation of pre-existing metabolic pathways. Triacylglycerol synthesis was increased only after a delay of 4 d, indicating the de novo induction of enzymes necessary for triacylglycerol metabolism. Retinol incorporation and conversion into retinyl esters were also considerably increased by previous incubation with retinoids. Retinoid-induced changes in GRX cells provide a model for studying in vitro the interconversion of liver connective tissue cells between the myofibroblastic and fat-storing phenotypes. This interconversion is considered to be one of the major control points of normal homeostasis and of pathological modifications of liver connective tissue.

The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast.

Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV) is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22). Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited beta-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374.

[A technology with multiple applications]. / Uma tecnologia com múltiplas aplicações.

Plant breeding has been a human practice for some thousands of years. However, this process of domestication has made plants more vulnerable to pests and diseases. Classic plant breeding has allowed the genetic manipulation of plants through crossings with a resulting increase in crop productivity. Recently, the recombinant DNA technology has increased the possibilities of integration of exogenous genes to the plant genome, resulting in the production of transgenic plants. Despite the great debate on this issue, such plants represent to date a promising avenue for plant breeding. There are many examples of gene transference strategies which have been successful in promoting resistance to herbicides, viruses, fungi, bacteria and insects, or in producing an increase in food quality. In addition to biotechnological applications, transgenic plants have made a significant contribution to the study of gene functioning, such as the analysis of genic expression regulation and the study of protein functions codified by distinct plant genes.

Modulation of genes related to specific metabolic pathways in response to cytosolic ascorbate peroxidase knockdown in rice plants.

As a central component of the hydrogen peroxide detoxifying system in plant cells, ascorbate peroxidases (APX) play an essential role in the control of intracellular reactive oxygen species (ROS) levels. To characterise the function of cytosolic APX isoforms (OsAPX1 and OsAPX2) in the mechanisms of plant defence, OsAPX1/2 knockdown rice plants were previously obtained. OsAPX1/2 knockdown plants (APx1/2s) exhibited a normal phenotype and development, even though they showed a global reduction of APX activity and increased hydrogen peroxide accumulation. To understand how rice plants compensate for the deficiency of cytosolic APX, expression and proteomic analyses were performed to characterise the global expression pattern of the APx1/2s mutant line compared with non-transformed plants. Our results strongly suggest that deficiencies in cytosolic APX isoforms markedly alter expression of genes associated with several key metabolic pathways, especially of genes involved in photosynthesis and antioxidant defence. These metabolic changes are compensatory because central physiological processes such as photosynthesis and growth were similar to non-transformed rice plants. Our analyses showed modulation of groups of genes and proteins related to specific metabolic pathways. Among the differentially expressed genes, the largest number corresponded to those with catalytic activity. Genes related to oxidative stress, carbohydrate metabolism, photosynthesis and transcription factor-encoding genes were also modulated. These results represent an important step toward understanding of the role played by cytosolic APX isoforms and hydrogen peroxide in the regulation of metabolism by redox modulation in monocots.

Relationship between oxidative stress levels and activation state on a hepatic stellate cell line.

BACKGROUND/AIMS: Oxidative stress plays an important role in liver fibrosis. Under pathological conditions, hepatic stellate cells (HSC) undergo an activation process, developing a myofibroblast-like phenotype from the lipocyte phenotype. In this study, we determined the levels of oxidative stress and proliferation in different activation states of an experimental model of mouse HSC, the GRX cell line. These cells can be induced in vitro to display a more activated state or a quiescent phenotype. METHODS/RESULTS: We observed increased oxidative damage and higher levels of reactive oxygen species, measured by thiobarbituric acid reactive species and 2',7'-dichlorofluorescein diacetate, respectively, and diminished catalase activity in activated cells. Activation decreased proliferation and increased the number of cells in G2/M. Antioxidants N-acetylcysteine and Trolox varied in their capacity to correct the oxidative stress and proliferation status. CONCLUSIONS: The differences in physiological functions of stellate cell phenotypes suggest a relationship between oxidative stress levels and activation state.

Optimal sampling strategy for estimation of spatial genetic structure in tree populations.

Fine-scale spatial genetic structure (SGS) in natural tree populations is largely a result of restricted pollen and seed dispersal. Understanding the link between limitations to dispersal in gene vectors and SGS is of key interest to biologists and the availability of highly variable molecular markers has facilitated fine-scale analysis of populations. However, estimation of SGS may depend strongly on the type of genetic marker and sampling strategy (of both loci and individuals). To explore sampling limits, we created a model population with simulated distributions of dominant and codominant alleles, resulting from natural regeneration with restricted gene flow. SGS estimates from subsamples (simulating collection and analysis with amplified fragment length polymorphism (AFLP) and microsatellite markers) were correlated with the 'real' estimate (from the full model population). For both marker types, sampling ranges were evident, with lower limits below which estimation was poorly correlated and upper limits above which sampling became inefficient. Lower limits (correlation of 0.9) were 100 individuals, 10 loci for microsatellites and 150 individuals, 100 loci for AFLPs. Upper limits were 200 individuals, five loci for microsatellites and 200 individuals, 100 loci for AFLPs. The limits indicated by simulation were compared with data sets from real species. Instances where sampling effort had been either insufficient or inefficient were identified. The model results should form practical boundaries for studies aiming to detect SGS. However, greater sample sizes will be required in cases where SGS is weaker than for our simulated population, for example, in species with effective pollen/seed dispersal mechanisms.

Monitoring genetic diversity in tropical trees with multilocus dominant markers.

Since no universal codominant markers are currently available, dominant genetic markers, such as amplified fragment length polymorphism (AFLP), are valuable tools for assessing genetic diversity in tropical trees. However, the measurement of genetic diversity (H) with dominant markers depends on the frequency of null homozygotes (Q) and the fixation index (F) of populations. While Q can be estimated for AFLP loci, F is less accessible. Through a modelling approach, we show that the monolocus estimation of genetic diversity is strongly dependent on the value of F, but that the multilocus diversity estimate is surprisingly robust to variations in F. The robustness of the estimate is due to a mechanistic effect of compensation between negative and positive biases of H by different AFLP loci exhibiting contrasting frequency profiles of Q. The robustness was tested across contrasting theoretical frequency profiles of Q and verified for 10 neotropical species. Practical recommendations for the implementation of this analytical method are given for genetic surveys in tropical trees, where such markers are widely applied.

Effects of site-directed mutagenesis on the presumed catalytic triad and substrate-binding pocket of grapevine fanleaf nepovirus 24-kDa proteinase.

Grapevine fanleaf nepovirus (GFLV) has a bipartite plus-sense RNA genome. Its structural and functional proteins originate from polyprotein maturation by at least one virus-encoded proteinase. Here we describe the cloning of the 24-kDa proteinase cistron located between the virus-linked protein (VPg) and the RNA-dependent RNA polymerase cistron in GFLV RNA1 (nucleotides 3966 to 4622). Proteinase expressed from this clone is able to cleave GFLV polyprotein P2 in order to produce the coat protein and a 66-kDa protein which is further processed to the 38-kDa presumed movement protein. The GFLV 24-kDa proteinase sequence contains sequence similarities with other nepovirus and comovirus proteinases, particularly at the level of the conserved domains corresponding to the hypothetical catalytic triad and to the substrate-binding pocket (amino acids 192 to 200). Site-directed mutagenesis of residues His43, Glu87, and Leu197 abolished proteinase activity. Inactivation of the enzyme is also observed if the catalytic residue Cys179 was substituted by isoleucine, but replacement by a serine at the same position produced a mutant with an activity identical to that of native proteinase. All our data show that GFLV cysteine proteinase presents structure similarities to the proteinases of cowpea mosaic virus and potyviruses but is most closely related to trypsin.

Quantification of attached cells in tissue culture plates and on microcarriers.

A method for quantification of anchorage-dependent cells in culture on plane surfaces or on microcarriers is proposed. It is based on Coomassie brilliant blue R-250 adsorption, followed by elution of the dye and measurement by spectrophotometry at 595 nm. A linear correlation (r = 0.988 to 0.996) was observed between absorbance and cell number along a large range of cell densities. This technique may be used for monitoring cell growth, from seeding of initial inoculi to scaling up of cultures in bioreactors.

VPg Northern-immunoblots as a means for detection of viral RNAs in protoplasts or plants infected with grapevine fanleaf nepovirus.

Anti-genome-linked viral protein (anti-VPg) antibodies were produced from a synthetic peptide corresponding to the integral VPg sequence of grapevine fanleaf nepovirus-F13. These antibodies allowed detection of viral VPg-linked proteins which occur during the processing of viral polyproteins and of viral RNAs in total RNA extracts from infected protoplasts or plants after Northern blotting. These highly specific antibodies recognised RNAs from two grapevine fanleaf virus strains but not from arabis mosaic virus.

Cloning and in vitro characterization of the grapevine fanleaf virus proteinase cistron.

The region of the genomic RNA-1 from grapevine fanleaf virus isolate F13 (GFLV-F13), containing the proteinase cistron and flanking sequences (nucleotides 3894 to 4789) of the GFLV polyprotein, was modified by PCR mutagenesis to create a start codon and cloned in a transcription vector. The transcripts from the resulting clone (pVP7) produced, upon translation in rabbit reticulocyte lysate, a 37.8-kDa protein which was subsequently cleaved to a stable 28-kDa product. Autocleavage was maximal at pH 7.0-8.5 and at 30 degrees. Inhibition of the activity was greater than 80% when translation was performed in the wheat germ system. In rabbit reticulocyte lysate, inhibition was also obtained with PMSF, EDTA, E-64, Ca+2, Zn+2, and Co+2. The pVP7 translation product acts in cis, in the case of its autocleavage, or in trans in the processing of the viral 122-kDa polyprotein from GFLV RNA-2 into a 66-kDa protein and the 56-kDa coat protein. The carboxy extremity of the complete pVP7 translation product, encoded by nucleotides 4633 to 4789 of RNA-1, was not required for the proteinase activity, at least in trans.

Collagen synthesis in an established liver connective tissue cell line (GRX) during induction of the fat-storing phenotype.

We have studied collagen synthesis and secretion in an established liver connective tissue cell line (GRX) that can be induced in vitro to express either the myofibroblastic or the fat-storing (lipocyte) phenotype. In lipocytes, collagen synthesis was reduced. Their intracellular collagen degradation corresponded to 15% of newly synthesized collagen. In myofibroblasts, collagen synthesis was high but its secretion was considerably altered by intracellular collagen degradation, which attained up to 60% of newly synthesized collagen. In this in vitro model, we have provided direct evidence that hepatic lipocytes, involved mainly in lipid and retinol metabolism, have a low basal level of collagen synthesis. Myofibroblastic phenotype correlates with increased collagen synthesis and may be directly related to increased collagen deposition in hepatic fibrosis. Modulation of the phenotype of liver connective tissue cells are possibly one of the major points of control in normal and pathological deposition of collagen in liver parenchyma.

Structural and phylogenetic relationships among plant and animal cystatins.

The plant cystatins or phytocystatins (PhyCys) are cysteine proteinase inhibitors containing the QxVxG motif and have been placed in the cystatin superfamily of proteins. The primary sequences of PhyCys have a high degree of homology with the members of the cystatin family, but they resemble stefins by the absence of disulfide bonds and cysteine residues. A multialignment and a phylogenetic analysis of 63 cystatins, 32 of which are PhyCys, demonstrate that all PhyCys cluster in a major evolutionary tree branch and support the classification of PhyCys as a new cystatin family. The PhyCys also possess a specific consensus sequence [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ] -N placed on the region corresponding to a predictable amino-terminal alpha-helix. This sequence can be used to specifically identify PhyCys on protein data banks and to differentiate them from the other members of the superfamily.

Genome organization of grapevine fanleaf nepovirus RNA2 deduced from the 122K polyprotein P2 in vitro cleavage products.

The full-length transcript of grapevine fanleaf virus (GFLV) RNA2 produces a primary product of 122K when translated in the rabbit reticulocyte system. This 122K polyprotein is completely processed in vitro by the RNA1-encoded 24K proteinase. The positions of the cleavage sites within the polyprotein have been mapped and the genome organization of GFLV-F13 RNA2 has been established. The order of mature proteins in the 122K polyprotein is the amino-terminal 28K protein, the 38K protein followed by the 56K coat protein at the carboxy terminus. These proteins represent the final cleavage products of the 122K polyprotein. A 66K protein which yields 28K and 38K proteins constitutes the major maturation intermediate. Microsequencing of the amino extremity of radioactively labelled 38K protein allowed identification of the Cys257/Ala258 site as the cleavage site recognized by the GFLV proteinase between the 28K and the 38K proteins in the 66K protein in addition to the Arg605/Gly606 site between the 38K protein and the coat protein.

Effects of retinol on proliferation, cell adherence and extracellular matrix synthesis in a liver myofibroblast or lipocyte cell line (GRX).

We have studied the effect of retinol on an established murine cell line (GRX), representative of liver connective tissue cells. This cell line has myofibroblast characteristics; under retinol treatment it is induced into the lipocyte (Ito-cell) phenotype. Retinol decreased the proliferation rate in the entire cell population. It increased cell adherence to the substrate, which was correlated with the increased secretion of fibronectin. Collagen secretion was specifically decreased, whilst the total protein secretion remained stable. Heparan sulphate was decreased in the pericellular compartment, but other glycosaminoglycans were not affected by retinol treatment. Modulations of pericellular components induced by retinol may alter the relations among liver mesenchymal cells, and may be related to vitamin-A-induced modifications of the homoeostasis of hepatic connective tissue and hepatic fibrosis.

Retinol uptake and metabolism, and cellular retinol binding protein expression in an in vitro model of hepatic stellate cells.

Liver is a major site of retinoid metabolism and storage, and more than 80% of the liver retinoids are stored in hepatic stellate cells. These cells represent less than 1% of the total liver protein, reaching a very high relative intracellular retinoid concentration. The plasma level of retinol is maintained close to 2 microM, and hepatic stellate cells have to be able both to uptake or to release retinol depending upon the extracellular retinol status. In view of their paucity in the liver tissue, stellate cells have been studied in primary cultures, in which they loose rapidly the stored lipids and retinol, and convert spontaneously into the activated myofibroblast phenotype, turning a long-term study of their retinol metabolism impossible. We have analyzed the retinol metabolism in the established GRX cell line, representative of stellate cells. We showed that this cell line behaves very similarly, with respect the retinol uptake and release, to primary cultures of hepatic stellate cells. Moreover, we showed that the cellular retinol binding protein (CRBP-I) expression in these cells, relevant for both uptake and esterification of retinol, responds to the extracellular retinol status, and is correlated to the retinol binding capacity of the cytosol. Its expression is not associated with the overall induction of the lipocyte phenotype by other agents. We conclude that the GRX cell line represents an in vitro model of hepatic stellate cells, and responds very efficiently to wide variations of the extracellular retinol status by autonomous controls of its uptake, storage or release.