Reduced acute functional tolerance and enhanced preference for ethanol in Caenorhabditis elegans exposed to lead during development: Potential role of alcohol dehydrogenase.

Despite its relative simplicity, the invertebrate Caenorhabditis elegans (C. elegans) has become a powerful tool to evaluate toxicity. Lead (Pb) persistence in the environment and its distinctive characteristic as a neurodevelopmental toxicant determine the potential effects of this metal against challenging events later in life. Additionally, among other psychoactive substances, low to moderate ethanol (EtOH) doses have been pointed out to induce behaviors such as acute functional tolerance (AFT) and drug-induced chemotaxis. In the present study, we aimed to study the impact of early-life Pb exposure on EtOH-induced motivational and stimulant effects in C. elegans by assessing the preference for EtOH and the participation of alcohol dehydrogenase (ADH, sorbitol dehydrogenase -SODH in worms) in the AFT response. Thus, N2 (wild type) and RB2114 (sod-1 -/-) strains developmentally exposed to 24 µM Pb were evaluated in their AFT to 200 mM EtOH alone and in combination with acetaldehyde (ACD). We ascribed the enhanced EtOH-induced AFT observed in the N2 Pb-exposed animals to a reduced ADH functionality as evaluated by both, ADH activity determination and the allyl alcohol test, which altogether suggest excess EtOH accumulation rather than low ACD formation in these animals. Moreover, the Pb-induced preference for EtOH indicates enhanced motivational effects of this drug as a consequence of early-life exposure to Pb, results that resemble our previous reports in rodents and provide a close association between EtOH stimulant and motivational effects in these animals.

Assessment and characterization of tomato lipophilic electrophiles and their potential contribution to nutraceutical properties via SKN-1/Nrf2 signaling activation.

Phytochemical electrophiles are drawing significant attention due to their properties to modulate signaling pathways related to cellular homeostasis. The aim of this study was to develop new tools to examine the electrophilic activity in food and predict their beneficial effects on health. We developed a spectrophotometric assay based on the nitrobenzenethiol (NBT) reactivity, as a thiol-reactive nucleophile, to screen electrophiles in tomato fruits. The method is robust, simple, inexpensive, and could be applied to other types of food. We quantified the electrophile activity in a tomato collection and associated this activity with the pigment composition. Thus, we identified lycopene, ß- and γ-carotenes, 16 by-products of carotenoid oxidation and 18 unknown compounds as NBT-reactive by HPLC-MS/MS. The potential benefits of NBT-reactive compounds on health were evaluated in the in vivo model of C. elegans where they activated the SKN-1/Nrf2 pathway, evidencing the ability of electrophilic compounds to induce a biological response.

The cytosolic invertase NI6 affects vegetative growth, flowering, fruit set, and yield in tomato.

Sucrose metabolism is important for most plants, both as the main source of carbon and via signaling mechanisms that have been proposed for this molecule. A cleaving enzyme, invertase (INV) channels sucrose into sink metabolism. Although acid soluble and insoluble invertases have been largely investigated, studies on the role of neutral invertases (A/N-INV) have lagged behind. Here, we identified a tomato A/N-INV encoding gene (NI6) co-localizing with a previously reported quantitative trait locus (QTL) largely affecting primary carbon metabolism in tomato. Of the eight A/N-INV genes identified in the tomato genome, NI6 mRNA is present in all organs, but its expression was higher in sink tissues (mainly roots and fruits). A NI6-GFP fusion protein localized to the cytosol of mesophyll cells. Tomato NI6-silenced plants showed impaired growth phenotype, delayed flowering and a dramatic reduction in fruit set. Global gene expression and metabolite profile analyses of these plants revealed that NI6 is not only essential for sugar metabolism, but also plays a signaling role in stress adaptation. We also identified major hubs, whose expression patterns were greatly affected by NI6 silencing; these hubs were within the signaling cascade that coordinates carbohydrate metabolism with growth and development in tomato.

Validated MAGIC and GWAS population mapping reveals the link between vitamin E content and natural variation in chorismate metabolism in tomato.

Tocochromanols constitute the different forms of vitamin E (VTE), essential components of the human diet, and display a high membrane protectant activity. By combining interval mapping and genome-wide association studies (GWAS), we unveiled the genetic determinants of tocochromanol accumulation in tomato (Solanum lycopersicum) fruits. To enhance the nutritional value of this highly consumed vegetable, we dissected the natural intraspecific variability of tocochromanols in tomato fruits and genetically engineered their biosynthetic pathway. These analyses allowed the identification of a total of 25 quantitative trait loci interspersed across the genome pinpointing the chorismate-tyrosine pathway as a regulatory hub controlling the supply of the aromatic head group for tocochromanol biosynthesis. To validate the link between the chorismate-tyrosine pathway and VTE, we engineered tomato plants to bypass the pathway at the arogenate branch point. Transgenic tomatoes showed moderate increments in tocopherols (up to approximately 20%) and a massive accumulation of tocotrienols (up to approximately 3400%). Gene expression analyses of these plants reveal a trade-off between VTE and natural variation in chorismate metabolism explained by transcriptional reprogramming of specific structural genes of the pathway. By restoring the accumulation of alpha-tocotrienols (α-t3) in fruits, the plants produced here are of high pharmacological and nutritional interest.

Effects of chlorogenic acid on thermal stress tolerance in C. elegans via HIF-1, HSF-1 and autophagy.

BACKGROUND: Chlorogenic acid (CGA) is a polyphenol widely distributed in plants and plant-derived food with antioxidant and protective activities against cell stress. Caenorhabditis elegans is a model organism particularly useful for understanding the molecular and biochemical mechanisms associated with aging and stress in mammals. In C. elegans, CGA was shown to improve resistance to thermal, while the underlying mechanisms that lead to this effect require further understanding. PURPOSE: The present study was conducted to investigate the underlying molecular mechanisms behind CGA response conferring thermotolerance to C. elegans. METHODS AND RESULTS: Signaling pathways that could be involved in the CGA-induced thermotolerance were evaluated in C. elegans strains with loss-of-function mutation. CGA-induced thermotolerance required hypoxia-inducible factor HIF-1 but no insulin pathway. CGA exposition (1.4 µM CGA for 18 h) before thermal stress treatment increased HIF-1 levels and activity. HIF-1 activation could be partly attributed to an increase in radical oxygen species and a decrease in superoxide dismutase activity. In addition, CGA exposition before thermal stress also increased autophagy just as hormetic heat condition (HHC), worms incubated at 36 °C for 1 h. RNAi experiments evidenced that autophagy was increased by CGA via HIF-1, heat-shock transcription factor HSF-1 and heat-shock protein HSP-16 and HSP-70. In contrast, autophagy induced by HHC only required HSF-1 and HSP-70. Moreover, suppression of autophagy induction showed the significance of this process for adapting C. elegans to cope with thermal stress. CONCLUSION: This study demonstrates that CGA-induced thermotolerance in C. elegans is mediated by HIF-1 and downstream, by HSF-1, HSPs and autophagy resembling HHC.

A Tomato Tocopherol-Binding Protein Sheds Light on Intracellular α-Tocopherol Metabolism in Plants.

Tocopherols are non-polar compounds synthesized in the plastids, which function as major antioxidants of the plant cells and are essential in the human diet. Both the intermediates and final products of the tocopherol biosynthetic pathway must cross plastid membranes to reach their sites of action. So far, no protein with tocopherol binding activity has been reported in plants. Here, we demonstrated that the tomato SlTBP protein is targeted to chloroplasts and able to bind α-tocopherol. SlTBP-knockdown tomato plants exhibited reduced levels of tocopherol in both leaves and fruits. Several tocopherol deficiency phenotypes were apparent in the transgenic lines, such as alterations in photosynthetic parameters, dramatic distortion of thylakoid membranes and significant variations in the lipid profile. These results, along with the altered expression of genes related to photosynthesis, and tetrapyrrole, lipid, isoprenoid, inositol/phosphoinositide and redox metabolism, suggest that SlTBP may act in conducting tocopherol (or its biosynthetic intermediates) between the plastid compartments and/or at the interface between chloroplast and endoplasmic reticulum membranes, affecting interorganellar lipid metabolism.

Contrasting metabolic profiles of tasty Andean varieties of tomato fruit in comparison with commercial ones.

BACKGROUND: The fruits of most commercial tomato cultivars (Solanum lycopersicum L.) are deficient in flavour. In contrast, traditional 'criollo' tomato varieties are appreciated for fruit of excellent organoleptic quality. Small farmers from the Andean valleys in Argentina have maintained their own tomato varieties, which were selected mainly for flavour. This work aims to correlate the chemical composition of the fruit with the sensory attributes of eight heirloom tomato varieties. The long-term goal is to identify potential candidate genes capable of altering the chemicals involved in flavour. RESULTS: A sensory analysis was conducted and the metabolomics of fruit were determined. The data revealed that defined tomato aroma and sourness correlated with citrate and several volatile organic compounds (VOC), such as α-terpineol, p-menth-1-en-9-al, linalool and 3,6-dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran (DMHEX), a novel volatile recently identified in tomato. Two sensory attributes - sweetness and a not-acidic taste - correlated with the characteristic tomato taste, and also with fructose, glucose, and two VOCs, benzaldehyde, and 2-methyl-2-octen-4-one. CONCLUSIONS: These data provide new evidence of the complex chemical combination that induced the flavour and aroma of the good-tasting 'criollo' tomato fruit. That is, the compounds that correlated with defined tomato aroma and acidic taste did not correlate with sweetness, or with characteristic tomato taste. © 2018 Society of Chemical Industry.

Neuronal network analyses reveal novel associations between volatile organic compounds and sensory properties of tomato fruits.

INTRODUCTION: The process of tomato (Solanum lycopersicum) breeding has affected negatively the fruit organoleptic properties and this is evident when comparing modern cultivars with heirloom varieties. Flavor of tomato fruit is determined by a complex combination of volatile and nonvolatile metabolites that is not yet understood. OBJECTIVES: The aim of this work was to provide an alternative approach to exploring the relationship between tomato odour/taste and volatile organic compounds (VOCs). METHODS: VOC composition and organoleptic properties of seven Andean tomato landraces along with an edible wild species (Solanum pimpinellifolium) and four commercial varieties were characterized. Six hedonic traits were analyzed by a semitrained sensory panel to describe the organoleptic properties. Ninety-four VOCs were analyzed by headspace solid phase microextraction/gas chromatography-mass spectrometry (HS/SPME/GC-MS). The relationship between sensory data and VOCs was explored using an Artificial Neural Networks model (Kohonen Self Organizing Maps, omeSOM). RESULTS AND CONCLUSION: The results showed a strong preference by panelists for tomatoes of landraces than for commercial varieties and wild species. The predictive analysis by omeSOM showed 15 VOCs significantly associated to the typical and atypical tomato odour and taste. Moreover, omeSOM was used to predict the relationship of VOC ratios with sensory data. A total of 108 VOC ratios out of 8837 VOC ratios were predicted to be contributing to the typical and atypical tomato odour and taste. The metabolic origin of these flavor-associated VOCs and the metabolic point or target for breeding strategies were discussed.

Peanut Seed Cultivars with Contrasting Resistance to Aspergillus parasiticus Colonization Display Differential Temporal Response of Protease Inhibitors.

Significant efforts are being made to minimize aflatoxin contamination in peanut seeds and one possible strategy is to understand and exploit the mechanisms of plant defense against fungal infection. In this study we have identified and characterized, at biochemical and molecular levels, plant protease inhibitors (PPIs) produced in peanut seeds of the resistant PI 337394 and the susceptible Forman cultivar during Aspergillus parasiticus colonization. With chromatographic methods and 2D-electrophoresis-mass spectrometry we have isolated and identified four variants of Bowman-Birk trypsin inhibitor (BBTI) and a novel Kunitz-type protease inhibitor (KPI) produced in response to A. parasiticus colonization. KPI was detected only in the resistant cultivar, while BBTI was produced in the resistant cultivar in a higher concentration than susceptible cultivar and with different isoforms. The kinetic expression of KPI and BBTI genes along with trypsin inhibitory activity was analyzed in both cultivars during infection. In the susceptible cultivar an early PPI activity response was associated with BBTI occurrence. Meanwhile, in the resistant cultivar a later response with a larger increase in PPI activity was associated with BBTI and KPI occurrence. The biological significance of PPI in seed defense against fungal infection was analyzed and linked to inhibitory properties on enzymes released by the fungus during infection, and to the antifungal effect of KPI.

Sensitive biomarker responses of the shrimp Palaemonetes argentinus exposed to chlorpyrifos at environmental concentrations: Roles of alpha-tocopherol and metallothioneins.

The aim of this study was to evaluate the toxic effects of chlorpyrifos (CPF) at environmental concentrations on the shrimp Palaemonetes argentinus, a South American native species. Organisms were exposed to environmentally relevant concentrations of CPF (from 3.5 to 94.5ngCPFL(-1)) at laboratory conditions for 96h. A wide battery of biochemical responses including bioaccumulation, damage and defense biomarkers were measured in cephalothorax and abdomen of shrimp. The concentration of CPF was below the detection limit of the method in both body sectors (8ngCPFg(-1)ww), probably indicating fast biotransformation of the parental compound. Our results showed that CPF exposure inhibits acetylcholinesterase activity from 3.5ngCPFL(-1), a concentration below the suggested Argentinean guidelines for the protection of aquatic biota. Moreover, oxidative stress was evidenced by increased H2O2 content and increased levels of TBARs and carbonyl groups in proteins. The induction of antioxidant enzymes like catalase, glutathione S-transferase and glutathione peroxidase seems not be sufficient to prevent oxidative damages. In addition, the mobilization of α-tocopherol from abdomen to cephalothorax was observed and reported for the first time in non-reproductive condition. Likewise, a strong diminution of metallothioneins occurred in cephalothorax from the lowest CPF concentration while induction occurred from the same treatment in abdomen as an oxidative stress response. Finally, significant correlation between Integrated Biomarker Response values and exposure concentrations suggest the usefulness of P. argentinus as bioindicator of CPF exposure at concentrations as low as environmental ones.

Data on polyphenols and biological activity analyses of an Andean tomato collection and their relationships with tomato traits and geographical origin.

Data provide information about a tomato collection composed of accessions from the Andean Valley, commercial accessions and wild species. Antioxidant metabolites were measured in mature fruits of this collection, and their biological activities were assessed by both in vitro and in vivo methods. In this work, the parameters used to identify and quantify polyphenols compounds in tomato fruit by liquid chromatography coupled to diode array detector and quadrupole time of flight mass spectrometer are described. Moreover, data supporting a procedure to characterize the properties of tomato fruits to revert death by thermal stress in Caenorhabditis elegans are explained in detail. Lastly, principal component analysis and hierarchical cluster analysis of metabolites composition, antioxidant activities (in vivo and in vitro), tomato traits and geographical origin of the tomatoes collection are shown. The data presented here are related to the research article entitled "Hydrophilic antioxidants from Andean Tomato Landraces assessed by their bioactivities in vitro and in vivo" [1].

Hydrophilic antioxidants from Andean tomato landraces assessed by their bioactivities in vitro and in vivo.

Potential nutraceutical properties of hydrophilic antioxidants in fruits of tomato landraces collected in Andean valleys were characterised. Antioxidant metabolites were measured by HPLC-DAD-MS/MS in mature fruits and their biological activities were assessed by in vitro and in vivo methods. In vitro antioxidant capacities were established by TEAC and FRAP methods. For in vivo biological activities we used a procedure based on Caenorhabditis elegans subjected to thermal stress. In addition, Saccharomyces cerevisiae was also used as a rapid screening system to evaluate tomato antioxidant capacity. All tomato accessions displayed significant differences regarding metabolic composition, biological activity and antioxidant capacity. Metabolite composition was associated with geographical origin and fruit size. Antioxidant activities showed significant association with phenolic compounds, such as caffeoylquinic acids, ferulic acid-O-hexosides and rutin. Combination of in vitro and in vivo methods applied here allowed evaluation of the variability in nutraceutical properties of tomato landraces, which could be applied to other fruits or food products.

Down-regulation of tomato PHYTOL KINASE strongly impairs tocopherol biosynthesis and affects prenyllipid metabolism in an organ-specific manner.

Tocopherol, a compound with vitamin E (VTE) activity, is a conserved constituent of the plastidial antioxidant network in photosynthetic organisms. The synthesis of tocopherol involves the condensation of an aromatic head group with an isoprenoid prenyl side chain. The latter, phytyl diphosphate, can be derived from chlorophyll phytol tail recycling, which depends on phytol kinase (VTE5) activity. How plants co-ordinate isoprenoid precursor distribution for supplying biosynthesis of tocopherol and other prenyllipids in different organs is poorly understood. Here, Solanum lycopersicum plants impaired in the expression of two VTE5-like genes identified by phylogenetic analyses, named SlVTE5 and SlFOLK, were characterized. Our data show that while SlFOLK does not affect tocopherol content, the production of this metabolite is >80% dependent on SlVTE5 in tomato, in both leaves and fruits. VTE5 deficiency greatly impacted lipid metabolism, including prenylquinones, carotenoids, and fatty acid phytyl esters. However, the prenyllipid profile greatly differed between source and sink organs, revealing organ-specific metabolic adjustments in tomato. Additionally, VTE5-deficient plants displayed starch accumulation and lower CO2 assimilation in leaves associated with mild yield penalty. Taken together, our results provide valuable insights into the distinct regulation of isoprenoid metabolism in leaves and fruits and also expose the interaction between lipid and carbon metabolism, which results in carbohydrate export blockage in the VTE5-deficient plants, affecting tomato fruit quality.

Fruits from ripening impaired, chlorophyll degraded and jasmonate insensitive tomato mutants have altered tocopherol content and composition.

Since isoprenoids are precursors in chlorophyll, carotenoid and tocopherol pathways, the study of their metabolism is of fundamental importance in understanding the regulatory cross-talk that contributes to the nutritional quality of tomato fruits. By means of an integrated analysis of metabolite and gene expression profiles, isoprenoid metabolism was dissected in ripening-impaired (ripening inhibitor and non-ripening), senescence-related (lutescent1 and green flesh) and jasmonate insensitive (jasmonic acid insensitive 1-1) tomato mutants, all in the Micro-Tom genetic background. It was found that the more upstream the location of the mutated gene, the more extensive the effect on the transcriptional profiles of the isoprenoid-related genes. Although there was a distinct effect in the analyzed mutations on chlorophyll, carotenoid and tocopherol metabolism, a metabolic adjustment was apparent such the antioxidant capacity mostly remained constant. Transcriptional profiles from fruits of ripening and senescence-related tomato mutants suggested that maintenance of the de novo phytyl diphosphate synthesis might, in later ripening stages, compensate for the lack of chlorophyll-derived phytol used in tocopherol production. Interestingly, an impairment in jasmonate perception led to higher total tocopherol levels in ripe fruits, accompanied by an increase in antioxidant capacity, highlighting the contribution of tocopherols to this nutritionally important trait.

Oxidative stress response induced by atrazine in Palaemonetes argentinus: the protective effect of vitamin E.

The widespread contamination and persistence of the herbicide atrazine residues in the environment resulted in the exposure of non-target organisms. The present study was undertaken to investigate the effect of atrazine in the response of oxidative stress biomarkers in the freshwater shrimp Palaemonetes argentinus and the protective effect of vitamin-E against atrazine-induced toxicity. Therefore, two batches of P. argentinus were fed for 21 days with a commercial food enriched in proteins (D1) or with D2, composed of D1 enriched with vitamin-E (6.8 and 16.0mg% of vitamin-E, respectively). Subsequently, half of the individuals of each group were exposed to atrazine (0.4mgL(-1)) for 24h and the others remained as controls. Atrazine promoted oxidative stress response in P. argentinus fed with D1 as indicated by enhanced H2O2 content and induction of superoxide dismutase, glutathione-S-transferases and glutathione reductase. This antioxidant activity would prevent the increment of thiobarbituric acid reactive substances in the shrimp tissues. P. argentinus fed with D2 reversed the response of the biomarkers measured. However, the activation of antioxidants response had an energetic cost, which was revealed by a decrease in lipids storage in shrimps. These results show the modulatory effect of vit-E on oxidative stress and its potential use as an effective antioxidant to be applied in chemoprotection strategies during aquaculture.

Natural occurring epialleles determine vitamin E accumulation in tomato fruits.

Vitamin E (VTE) content is a low heritability nutritional trait for which the genetic determinants are poorly understood. Here, we focus on a previously detected major tomato VTE quantitative trait loci (QTL; mQTL(9-2-6)) and identify the causal gene as one encoding a 2-methyl-6-phytylquinol methyltransferase (namely VTE3(1)) that catalyses one of the final steps in the biosynthesis of γ- and α-tocopherols, which are the main forms of VTE. By reverse genetic approaches, expression analyses, siRNA profiling and DNA methylation assays, we demonstrate that mQTL(9-2-6) is an expression QTL associated with differential methylation of a SINE retrotransposon located in the promoter region of VTE3(1). Promoter DNA methylation can be spontaneously reverted leading to different epialleles affecting VTE3(1) expression and VTE content in fruits. These findings indicate therefore that naturally occurring epialleles are responsible for regulation of a nutritionally important metabolic QTL and provide direct evidence of a role for epigenetics in the determination of agronomic traits.

Silencing of the tomato sugar partitioning affecting protein (SPA) modifies sink strength through a shift in leaf sugar metabolism.

Limitations in our understanding about the mechanisms that underlie source-sink assimilate partitioning are increasingly becoming a major hurdle for crop yield enhancement via metabolic engineering. By means of a comprehensive approach, this work reports the functional characterization of a DnaJ chaperone related-protein (named as SPA; sugar partition-affecting) that is involved in assimilate partitioning in tomato plants. SPA protein was found to be targeted to the chloroplast thylakoid membranes. SPA-RNAi tomato plants produced more and heavier fruits compared with controls, thus resulting in a considerable increment in harvest index. The transgenic plants also displayed increased pigment levels and reduced sucrose, glucose and fructose contents in leaves. Detailed metabolic and enzymatic activities analyses showed that sugar phosphate intermediates were increased while the activity of phosphoglucomutase, sugar kinases and invertases was reduced in the photosynthetic organs of the silenced plants. These changes would be anticipated to promote carbon export from foliar tissues. The combined results suggested that the tomato SPA protein plays an important role in plastid metabolism and mediates the source-sink relationships by affecting the rate of carbon translocation to fruits.

Transcriptional regulation of tocopherol biosynthesis in tomato.

Tocopherols, compounds with vitamin E (VTE) activity, are potent lipid-soluble antioxidants synthesized only by photosynthetic organisms. Their biosynthesis requires the condensation of phytyl-diphosphate and homogentisate, derived from the methylerythritol phosphate (MEP) and shikimate pathways (SK), respectively. These metabolic pathways are central in plant chloroplast metabolism and are involved in the biosynthesis of important molecules such as chlorophyll, carotenoids, aromatic amino-acids and prenylquinones. In the last decade, few studies have provided insights into the regulation of VTE biosynthesis and its accumulation. However, the pathway regulatory mechanism/s at mRNA level remains unclear. We have recently identified a collection of tomato genes involved in tocopherol biosynthesis. In this work, by a dedicated qPCR array platform, the transcript levels of 47 genes, including paralogs, were determined in leaves and across fruit development. Expression data were analyzed for correlation with tocopherol profiles by coregulation network and neural clustering approaches. The results showed that tocopherol biosynthesis is controlled both temporally and spatially however total tocopherol content remains constant. These analyses exposed 18 key genes from MEP, SK, phytol recycling and VTE-core pathways highly associated with VTE content in leaves and fruits. Moreover, genomic analyses of promoter regions suggested that the expression of the tocopherol-core pathway genes is trancriptionally coregulated with specific genes of the upstream pathways. Whilst the transcriptional profiles of the precursor pathway genes would suggest an increase in VTE content across fruit development, the data indicate that in the M82 cultivar phytyl diphosphate supply limits tocopherol biosynthesis in later fruit stages. This is in part due to the decreasing transcript levels of geranylgeranyl reductase (GGDR) which restricts the isoprenoid precursor availability. As a proof of concept, by analyzing a collection of Andean landrace tomato genotypes, the role of the pinpointed genes in determining fruit tocopherol content was confirmed. The results uncovered a finely tuned regulation able to shift the precursor pathways controlling substrate influx for VTE biosynthesis and overcoming endogenous competition for intermediates. The whole set of data allowed to propose that 1-deoxy-D-xylulose-5-phosphate synthase and GGDR encoding genes, which determine phytyl-diphosphate availability, together with enzyme encoding genes involved in chlorophyll-derived phytol metabolism appear as the most plausible targets to be engineered aiming to improve tomato fruit nutritional value.

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.