Coupling Virus-Induced Gene Silencing to Exogenous Green Fluorescence Protein Expression Provides a Highly Efficient System for Functional Genomics in Arabidopsis and across All Stages of Tomato Fruit Development

Since the advent of the postgenomic era, efforts have focused on the development of rapid strategies for annotating plant genes of unknown function. Given its simplicity and rapidity, virus-induced gene silencing (VIGS) has become one of the preeminent approaches for functional analyses. However, several problems remain intrinsic to the use of such a strategy in the study of both metabolic and developmental processes. The most prominent of these is the commonly observed phenomenon of ""sectoring"" the tissue regions that are not effectively targeted by VIGS. To better discriminate these sectors, an effective marker system displaying minimal secondary effects is a prerequisite. Utilizing a VIGS system based on the tobacco rattle virus vector, we here studied the effect of silencing the endogenous phytoene desaturase gene (pds) and the expression and subsequent silencing of the exogenous green fluorescence protein (gfp) on the metabolism of Arabidopsis (Arabidopsis thaliana) leaves and tomato (Solanum lycopersicum) fruits. In leaves, we observed dramatic effects on primary carbon and pigment metabolism associated with the photobleached phenotype following the silencing of the endogenous pds gene. However, relatively few pleiotropic effects on carbon metabolism were observed in tomato fruits when pds expression was inhibited. VIGS coupled to gfp constitutive expression revealed no significant metabolic alterations after triggering of silencing in Arabidopsis leaves and a mild effect in mature green tomato fruits. By contrast, a wider impact on metabolism was observed in ripe fruits. Silencing experiments with an endogenous target gene of interest clearly demonstrated the feasibility of cosilencing in this system; however, carefully constructed control experiments are a prerequisite to prevent erroneous interpretation.

Genetic dissection of vitamin E biosynthesis in tomato

Vegetables are critical for human health as they are a source of multiple vitamins including vitamin E (VTE). In plants, the synthesis of VTE compounds, tocopherol and tocotrienol, derives from precursors of the shikimate and methylerythritol phosphate pathways. Quantitative trait loci (QTL) for alpha-tocopherol content in ripe fruit have previously been determined in an Solanum pennellii tomato introgression line population. In this work, variations of tocopherol isoforms (alpha, beta, gamma, and delta) in ripe fruits of these lines were studied. In parallel all tomato genes structurally associated with VTE biosynthesis were identified and mapped. Previously identified VTE QTL on chromosomes 6 and 9 were confirmed whilst novel ones were identified on chromosomes 7 and 8. Integrated analysis at the metabolic, genetic and genomic levels allowed us to propose 16 candidate loci putatively affecting tocopherol content in tomato. A comparative analysis revealed polymorphisms at nucleotide and amino acid levels between Solanum lycopersicum and S. pennellii candidate alleles. Moreover, evolutionary analyses showed the presence of codons evolving under both neutral and positive selection, which may explain the phenotypic differences between species. These data represent an important step in understanding the genetic determinants of VTE natural variation in tomato fruit and as such in the ability to improve the content of this important nutriceutical.

Genomic Analysis of Wild Tomato Introgressions Determining Metabolism- and Yield-Associated Traits

With the aim of determining the genetic basis of metabolic regulation in tomato fruit, we constructed a detailed physical map of genomic regions spanning previously described metabolic quantitative trait loci of a Solanum pennellii introgression line population. Two genomic libraries from S. pennellii were screened with 104 colocated markers from five selected genomic regions, and a total of 614 bacterial artificial chromosome (BAC)/cosmids were identified as seed clones. Integration of sequence data with the genetic and physical maps of Solanum lycopersicum facilitated the anchoring of 374 of these BAC/cosmid clones. The analysis of this information resulted in a genome-wide map of a nondomesticated plant species and covers 10% of the physical distance of the selected regions corresponding to approximately 1% of the wild tomato genome. Comparative analyses revealed that S. pennellii and domesticated tomato genomes can be considered as largely colinear. A total of 1,238,705 bp from both BAC/cosmid ends and nine large insert clones were sequenced, annotated, and functionally categorized. The sequence data allowed the evaluation of the level of polymorphism between the wild and cultivated tomato species. An exhaustive microsynteny analysis allowed us to estimate the divergence date of S. pennellii and S. lycopersicum at 2.7 million years ago. The combined results serve as a reference for comparative studies both at the macrosyntenic and microsyntenic levels. They also provide a valuable tool for fine-mapping of quantitative trait loci in tomato. Furthermore, they will contribute to a deeper understanding of the regulatory factors underpinning metabolism and hence defining crop chemical composition.

Determination of carbaryl in tomato ""in natura"" using an amperometric biosensor based on the inhibition of acetylcholinesterase activity

This work reports the utilization of two methodologies for carbaryl determination in tomatoes. The measurements were carried out using an amperometric biosensor technique based on the inhibition of acetylcholinesterase activity due to carbaryl adsorption and a HPLC procedure. The electrochemical experiments were performed in 0.1 mol L-1 phosphate buffer solutions at pH 7.4 with an incubation time of 8 min. The analytical curve obtained in pure solutions showed excellent linearity in the 5.0 x 10(-5) to 75 x 10(-5) mol L-1 range, with the limit of detection at 0.4 x 10(-3) gL(-1). The application of such a methodology in tomato samples involved solely liquidising the samples, which were spiked with 6.0 x 10(-6) and 5.0 x 10(-5) mol L-1 carbaryl. Recovery in such samples presented values of 99.0 and 92.4%, respectively. In order to obtain a comparison, HPLC experiments were also conducted under similar conditions. However, the tomato samples have to be manipulated by an extraction procedure (MSPD), which yielded much lower recovery values (78.3 and 84.8%, respectively). On the other hand, the detection limit obtained was much lower than that for the biosensor, i.e., 3.2 x 10(-6) g L-1. Finally, the biosensor methodology was employed to analyze carbaryl directly inside the tomato, without any previous manipulation. In this case, the biosensor was immersed in the tomato pulp, which had previously been spiked with the pesticide for 8 min, removed and inserted in the electrochemical cell. A recovery of 83.4% was obtained, showing very low interference of the matrix constituents. (C) 2007 Elsevier B.V. All rights reserved.

Phase transitions of frozen camu-camu (Myrciaria dubia (HBK) McVaugh) pulp: Effect of cryostabilizer addition

Camu-camu is a tropical fruit with very high vitamin C content and commercialized as frozen pulp. Enthalpies of freezing, temperatures of the onset of ice melting, and glass transition temperatures of the maximally freeze-concentrated phase (T`(g)) of camu-camu pulp and of samples containing maltodextrin (DE20) and sucrose were measured by differential scanning calorimetry. Maltodextrin exhibited the largest freeze stabilization potential, increasing T`(g) from -58.2 degrees C (natural pulp) to -39.6 degrees C when 30% (w/w) maltodextrin DE 20 was added. Sucrose showed negligible effect on T`(g) but enhanced considerably the freezing point depression and less amount of ice was formed.

Supercritical CO(2) extraction of carotenoids from pitanga fruits (Eugenia uniflora L.)

Supercritical carbon dioxide (SC-CO(2)) extraction was employed to extract carotenoids from the freeze-dried pulp of pitanga fruits (Eugenia uniflora L.), an exotic fruit, rich in carotenoids and still little explored commercially. The SC-CO(2) extraction was carried out at two temperatures, 40 and 60 degrees C, and seven pressures, 100, 150, 200, 250, 300, 350 and 400 bar. The carotenoids were determined by high-performance liquid chromatography connected to photodiode array and mass spectrometry detectors. Lycopene, rubixanthin and P-cryptoxanthin were the main carotenoids present in the freeze-dried pitanga pulp, whereas beta-cryptoxanthin concentration was negligible in the SC-CO(2) extracts, for all the investigated state conditions. The maximum recovery of carotenoids was obtained at 60 degrees C and 250 bar, extracting 55% of the total carotenoid content, 74% of the rubixanthin and 78% of the lycopene from the pulp. Under these state conditions, the total carotenoid concentration in the extract was 5474 mu g/g, represented by 66% lycopene and 32% rubixanthin. The experimental state conditions produced different SC-CO(2) extracts with respect to the extraction yield and concentration of different carotenoids, indicating that the supercritical carbon dioxide was selective in the extraction of the pitanga carotenoids as a function of temperature and pressure. (C) 2008 Elsevier B.V. All rights reserved.

DYNAMIC AND STEADY-SHEAR RHEOLOGICAL PROPERTIES OF XANTHAN AND GUAR GUMS DISPERSED IN YELLOW PASSION FRUIT PULP (Passiflora edulis f. flavicarpa)

Yellow passion fruit pulp is unstable, presenting phase separation that can be avoided by the addition of hydrocolloids. For this purpose, xanthan and guar gum [0.3, 0.7 and 1.0% (w/w)] were added to yellow passion fruit pulp and the changes in the dynamic and steady-shear rheological behavior evaluated. Xanthan dispersions showed a more pronounced pseudoplasticity and the presence of yield stress, which was not observed in the guar gum dispersions. Cross model fitting to flow curves showed that the xanthan suspensions also had higher zero shear viscosity than the guar suspensions, and, for both gums, an increase in temperature led to lower values for this parameter. The gums showed different behavior as a function of temperature in the range of 5-35 degrees C. The activation energy of the apparent viscosity was dependent on the shear rate and gum concentration for guar, whereas for xanthan these values only varied with the concentration. The mechanical spectra were well described by the generalized Maxwell model and the xanthan dispersions showed a more elastic character than the guar dispersions, with higher values for the relaxation time. Xanthan was characterized as a weak gel, while guar presented a concentrated solution behavior. The simultaneous evaluation of temperature and concentration showed a stronger influence of the polysaccharide concentration on the apparent viscosity and the G` and G `` moduli than the variation in temperature.

Seed Cell Wall Storage Polysaccharides: Models to Understand Cell Wall Biosynthesis and Degradation

Cell wall storage polysaccharides (CWSPs) are found as the principal storage compounds in seeds of many taxonomically important groups of plants. These groups developed extremely efficient biochemical mechanisms to disassemble cell walls and use the products of hydrolysis for growth. To accumulate these storage polymers, developing seeds also contain relatively high activities of noncellulosic polysaccharide synthases and thus are interesting models to seek the discovery of genes and enzymes related to polysaccharide biosynthesis. CWSP systems offer opportunities to understand phenomena ranging from polysaccharide deposition during seed maturation to the control of source-sink relationship in developing seedlings. By studying polysaccharide biosynthesis and degradation and the consequences for cell and physiological behavior, we can use these models to develop future biotechnological applications.

Molecular characterization of nitrate reductase gene and its expression in the marine red alga Gracilaria tenuistipitata (Rhodophyta)

The enzyme nitrate reductase (NR) responsible for the conversion of nitrate to nitrite is considered to be the rate-limiting step in nitrogen assimilation. The economically important marine macroalga Gracilaria tenuistipitata presents a circadian oscillation in NR protein content and activity. In order to identify if the regulation of NR in G. tenuistipitata happens at transcriptional levels, the NR cDNA and gene were sequenced and the NR mRNA expression was studied. Analysis of the sequenced gene revealed absence of introns which is unusual for NR genes. The transcriptional profiling revealed a circadian rhythm for NR; furthermore, a rhythm was observed in constant light condition, suggesting a possible regulation by the biological clock at the mRNA levels for NR in G. tenuistipitata.

Optimization of a method for determination of phenolic acids in exotic fruits by capillary electrophoresis

In this work, the separation of nine phenolic acids (benzoic, caffeic, chlorogenic, p-coumaric, ferulic, gallic, protocatechuic, syringic, and vanillic acid) was approached by a 32 factorial design in electrolytes consisting of sodium tetraborate buffer(STB) in the concentration range of 10-50 mmol L(-1) and methanol in the volume percentage of 5-20%. Derringer`s desirability functions combined globally were tested as response functions. An optimal electrolyte composed by 50 mmol L(-1) tetraborate buffer at pH 9.2, and 7.5% (v/v) methanol allowed baseline resolution of all phenolic acids under investigation in less than 15 min. In order to promote sample clean up, to preconcentrate the phenolic fraction and to release esterified phenolic acids from the fruit matrix, elaborate liquid-liquid extraction procedures followed by alkaline hydrolysis were performed. The proposed methodology was fully validated (linearity from 10.0 to 100 mu g mL(-1), R(2) > 0.999: LOD and LOQ from 1.32 to 3.80 mu g mL(-1) and from 4.01 to 11.5 mu g mL(-1), respectively; intra-day precision better than 2.8% CV for migration time and 5.4% CV for peak area; inter-day precision better than 4.8% CV for migration time and 4.8-11% CV for peak area: recoveries from 81% to 115%) and applied successfully to the evaluation of phenolic contents of abiu-roxo (Chrysophyllum caimito), wild mulberry growing in Brazil (Morus nigra L.) and tree tomato (Cyphomandra betacea). Values in the range of 1.50-47.3 mu g g(-1) were found, with smaller amounts occurring as free phenolic acids. (C) 2009 Elsevier B.V. All rights reserved.