Biomedical subjects

Fernando Carrari

Publications and source records attributed to Fernando Carrari.

At least 19 recordsLinked to original sources

PubMed2007

ci21A/Asr1 expression influences glucose accumulation in potato tubers.

Asr genes are exclusively found in the genomes of higher plants. In many species, this gene family is expressed under abiotic stress conditions and during fruit ripening. The encoded proteins have nuclear localisation and consequently a transcription factor function has been suggested. Interestingly, yeast-one-hybrid experiments revealed that a grape ASR binds to the promoter of a hexose transporter gene (VvHT1). However, the role of these proteins in planta is still elusive. By using a reverse genetics approach in potato we found that modification of Asr1 expression has no incidence on the aerial phenotype of the plant but exerts a dramatic effect in tuber. Asr1 antisense potatoes displayed decreased tuber fresh weight whereas Asr1 overexpressors had a diminished number of tubers. Moreover, overexpression lines showed lower transcript levels of a plasma membrane hexose transporter and a concomitant decrease in glucose content in parenchyma cells of potato tubers. On the same hand glucose uptake rate was also reduced in one of the overexpressing lines. It thus seems likely that Asr1 is involved in the control of hexose uptake in heterotrophic organs. In addition, the transgenic plants were characterized by several other changes in steady state metabolite levels. Results presented here support a role for ci21A/Asr1 in glucose metabolism of potato tuber.

DNA Primers↗

PubMed2006

Integrated analysis of metabolite and transcript levels reveals the metabolic shifts that underlie tomato fruit development and highlight regulatory aspects of metabolic network behavior.

Tomato (Solanum lycopersicum) is a well-studied model of fleshy fruit development and ripening. Tomato fruit development is well understood from a hormonal-regulatory perspective, and developmental changes in pigment and cell wall metabolism are also well characterized. However, more general aspects of metabolic change during fruit development have not been studied despite the importance of metabolism in the context of final composition of the ripe fruit. In this study, we quantified the abundance of a broad range of metabolites by gas chromatography-mass spectrometry, analyzed a number of the principal metabolic fluxes, and in parallel analyzed transcriptomic changes during tomato fruit development. Metabolic profiling revealed pronounced shifts in the abundance of metabolites of both primary and secondary metabolism during development. The metabolite changes were reflected in the flux analysis that revealed a general decrease in metabolic activity during ripening. However, there were several distinct patterns of metabolite profile, and statistical analysis demonstrated that metabolites in the same (or closely related) pathways changed in abundance in a coordinated manner, indicating a tight regulation of metabolic activity. The metabolite data alone allowed investigations of likely routes through the metabolic network, and, as an example, we analyze the operational feasibility of different pathways of ascorbate synthesis. When combined with the transcriptomic data, several aspects of the regulation of metabolism during fruit ripening were revealed. First, it was apparent that transcript abundance was less strictly coordinated by functional group than metabolite abundance, suggesting that posttranslational mechanisms dominate metabolic regulation. Nevertheless, there were some correlations between specific transcripts and metabolites, and several novel associations were identified that could provide potential targets for manipulation of fruit compositional traits. Finally, there was a strong relationship between ripening-associated transcripts and specific metabolite groups, such as TCA-cycle organic acids and sugar phosphates, underlining the importance of the respective metabolic pathways during fruit development.

Carbon↗

PubMed2006

Molecular identification of an Arabidopsis S-adenosylmethionine transporter. Analysis of organ distribution, bacterial expression, reconstitution into liposomes, and functional characterization.

Despite much study of the role of S-adenosylmethionine (SAM) in the methylation of DNA, RNA, and proteins, and as a cofactor for a wide range of biosynthetic processes, little is known concerning the intracellular transport of this essential metabolite. Screening of the Arabidopsis (Arabidopsis thaliana) genome yielded two potential homologs of yeast (Saccharomyces cerevisiae) and human SAM transporters, designated as SAMC1 and SAMC2, both of which belong to the mitochondrial carrier protein family. The SAMC1 gene is broadly expressed at the organ level, although only in specialized tissues of roots with high rates of cell division, and appears to be up-regulated in response to wounding stress, whereas the SAMC2 gene is very poorly expressed in all organs/tissues analyzed. Direct transport assays with the recombinant and reconstituted SAMC1 were utilized to demonstrate that this protein displays a very narrow substrate specificity confined to SAM and its closest analogs. Further experiments revealed that SAMC1 was able to function in uniport and exchange reactions and characterized the transporter as highly active, but sensitive to physiologically relevant concentrations of S-adenosylhomocysteine, S-adenosylcysteine, and adenosylornithine. Green fluorescent protein-based cell biological analysis demonstrated targeting of SAMC1 to mitochondria. Previous proteomic analyses identified this protein also in the chloroplast inner envelope. In keeping with these results, bioinformatics predicted dual localization for SAMC1. These findings suggest that the provision of cytosolically synthesized SAM to mitochondria and possibly also to plastids is mediated by SAMC1 according to the relative demands for this metabolite in the organelles.

Amino Acid Sequence↗

PubMed2006

Evolutionary history of the Asr gene family.

The Asr gene family is widespread in higher plants. Most Asr genes are up-regulated under different environmental stress conditions and during fruit ripening. ASR proteins are localized in the nucleus and their likely function is transcriptional regulation. In cultivated tomato, we identified a novel fourth family member, named Asr4, which maps close to its sibling genes Asr1-Asr2-Asr3 and displays an unshared region coding for a domain containing a 13-amino acid repeat. In this work we were able to expand our previous analysis for Asr2 and investigated the coding regions of the four known Asr paralogous genes in seven tomato species from different geographic locations. In addition, we performed a phylogenetic analysis on ASR proteins. The first conclusion drawn from this work is that tomato ASR proteins cluster together in the tree. This observation can be explained by a scenario of concerted evolution or birth and death of genes. Secondly, our study showed that Asr1 is highly conserved at both replacement and synonymous sites within the genus Lycopersicon. ASR1 protein sequence conservation might be associated with its multiple functions in different tissues while the low rate of synonymous substitutions suggests that silent variation in Asr1 is selectively constrained, which is probably related to its high expression levels. Finally, we found that Asr1 activation under water stress is not conserved between Lycopersicon species.

Amino Acid Sequence↗

PubMed2006

Comprehensive metabolic profiling and phenotyping of interspecific introgression lines for tomato improvement.

Tomato represents an important source of fiber and nutrients in the human diet and is a central model for the study of fruit biology. To identify components of fruit metabolic composition, here we have phenotyped tomato introgression lines (ILs) containing chromosome segments of a wild species in the genetic background of a cultivated variety. Using this high-diversity population, we identify 889 quantitative fruit metabolic loci and 326 loci that modify yield-associated traits. The mapping analysis indicates that at least 50% of the metabolic loci are associated with quantitative trait loci (QTLs) that modify whole-plant yield-associated traits. We generate a cartographic network based on correlation analysis that reveals whole-plant phenotype associated and independent metabolic associations, including links with metabolites of nutritional and organoleptic importance. The results of our genomic survey illustrate the power of genome-wide metabolic profiling and detailed morphological analysis for uncovering traits with potential for crop breeding.

Computer Simulation↗

PubMed2006

Metabolic regulation underlying tomato fruit development.

The development and maturation of tomato fruits has received considerable attention because of both the uniqueness of such processes to the biology of plants and the importance of these fruits as a component of the human diet. Molecular and genetic analysis of fruit development, and especially ripening of fleshy fruits, has resulted in significant gains in knowledge over recent years. A large amount of knowledge has been gathered on ethylene biosynthesis and response, cell wall metabolism, and environmental factors, such as light, that impact ripening. Considerably less attention has been paid directly to the general metabolic shifts that underpin these responses. Given the vast complexity of fruit metabolism, the focus chosen for this review is on primary metabolites and those secondary metabolites that are important with respect to fruit quality. Here, recent advances in dissecting tomato metabolic pathways are reviewed. Also discussed are recent examples in which the combined application of metabolic and transcriptional profiling, aimed at identifying candidate genes for modifying metabolite contents, was used.

Carbon↗

PubMed2006

Conversion of MapMan to allow the analysis of transcript data from Solanaceous species: effects of genetic and environmental alterations in energy metabolism in the leaf.

The tomato microarray TOM1 offers the possibility to monitor the levels of several thousand transcripts in parallel. The microelements represented on this tomato microarray have been putatively assigned to unigenes, and organised in functional classes using the MapMan ontology (Thimm et al., 2004. Plant J. 37: 914-939). This ontology was initially developed for use with the Arabidopsis ATH1 array, has a low level of redundancy, and can be combined with the MapMan software to provide a biologically structured overview of changes of transcripts, metabolites and enzyme activities. Use of this application is illustrated using three case studies with published or novel TOM1 array data sets for Solanaceous species. Comparison of previously reported data on transcript levels in potato leaves in the middle of the day and the middle of the night identified coordinated changes in the levels of transcripts of genes involved in various metabolic pathways and cellular events. Comparison with diurnal changes of gene expression in Arabidopsis revealed common features, illustrating how MapMan can be used to compare responses in different organisms. Comparison of transcript levels in new experiments performed on the leaves of the cultivated tomato S. lycopersicum and the wild relative S. pennellii revealed a general decrease of levels of transcripts of genes involved in terpene and, phenylpropanoid metabolism as well as chorismate biosynthesis in the crop compared to the wild relative. This matches the recently reported decrease of the levels of secondary metabolites in the latter. In the third case study, new expression array data for two genotypes deficient in TCA cycle enzymes is analysed to show that these genotypes have elevated levels of transcripts associated with photosynthesis. This in part explains the previously documented enhanced rates of photosynthesis in these genotypes. Since the Solanaceous MapMan is intended to be a community resource it will be regularly updated on improvements in tomato gene annotation and transcript profiling resources.

Arabidopsis↗

PubMed2006

Sucrose transporter LeSUT1 and LeSUT2 inhibition affects tomato fruit development in different ways.

Sucrose transporters of higher plants belong to a large gene family. At least four different sucrose transporters are known in Solanaceous plants, although their function remains to be elucidated in detail. The isolation of LeSUT1 and LeSUT2from Lycopersicon esculentum has been described earlier. Whereas SUT1 is supposed to be the main phloem loader of sucrose in Solanaceae, the role of SUT2 remains a matter of debate. A transgenic approach was taken to evaluate the potential functions of SUT2/SUC3 proteins in sucrose transport or sensing. Expression of LeSUT1 and LeSUT2 was inhibited independently in transgenic tomato plants, using the antisense technique, in order to analyse their specific functions. Although the phloem-specific inhibition of LeSUT1 antisense plants showed a phenotype consistent with an essential role in phloem loading, constitutive LeSUT2 antisense inhibition exclusively affected tomato fruit and seed development. Neither LeSUT1, nor the LeSUT2 antisense plants were able to produce normal tomato fruits; however, it is likely that independent mechanisms underlie these phenomena. While phloem loading was blocked in LeSUT1 antisense plants, the fertility of fruits was reduced in LeSUT2 antisense plants. A detailed physiological analysis of these plants established a role for SUT2 in pollen tube growth and thus assigned a physiological role for SUT2.

Base Sequence↗

PubMed2005

Modulation of fructokinase activity of potato (Solanum tuberosum) results in substantial shifts in tuber metabolism.

Potato plants (Solanum tuberosum L. cvs Desiree and Record) transformed with sense and antisense constructs of a cDNA encoding the potato fructokinase StFK1 exhibited altered transcription of this gene, altered amount of protein and altered enzyme activities. Measurement of the maximal catalytic activity of fructokinase revealed a 2-fold variation in leaf (from 90 to 180% of wild type activity) and either a 10- or 30-fold variation in tuber (from 10 or 30% to 300% in Record and Desiree, respectively) activity. The comparative effect of the antisense construct in leaf and tuber tissue suggests that this isoform is only a minor contributor to the total fructokinase activity in the leaf but the predominant isoform in the tuber. Antisense inhibition of the fructokinase resulted in a reduced tuber yield; however, its overexpression had no impact on this parameter. The modulation of fructokinase activity had few, consistent effects on carbohydrate levels, with the exception of a general increase in glucose content in the antisense lines, suggesting that this enzyme is not important for the control of starch synthesis. However, when metabolic fluxes were estimated, it became apparent that the transgenic lines display a marked shift in metabolism, with the rate of redistribution of radiolabel to sucrose markedly affected by the activity of fructokinase. These data suggest an important role for fructokinase, acting in concert with sucrose synthase, in maintaining a balance between sucrose synthesis and degradation by a mechanism independent of that controlled by the hexose phosphate-mediated activation of sucrose phosphate synthase.

Carbohydrate Metabolism↗

PubMed2005

Fruit carbohydrate metabolism in an introgression line of tomato with increased fruit soluble solids.

A tomato line (IL9-2-5) of the cultivated species, Lycopersicon esculentum, carrying a 9 cM introgression from the wild species, Lycopersicon pennelli, produces fruit with high soluble solids content (Brix), an important determinant of fruit quality for processing. Two quantitative trait loci (QTLs) relating to fruit soluble solids content have been identified within the introgressed segment. One of these QTLs (PW-9-2-5) is silent under the growth conditions used in this study, while a second (Brix-9-2-5) has been shown to encode a fruit apoplastic invertase (Lin5) with altered kinetic properties. In this study, we have undertaken a detailed biochemical analysis of the introgression line to attempt to gain an understanding of the metabolic changes associated with increased fruit soluble solids. Increased Brix in ripe fruit was shown to be the result of increased sucrose and glucose, with a more minor contribution from aspartate and alanine. The introgression leads to a pronounced increase in apoplastic invertase activity in the columella tissue that extends throughout fruit development. Furthermore, columella tissue from IL9-2-5 fruit has a greater capacity to take up exogenously supplied sucrose, an observation that is consistent with the kinetic properties of the introgressed Lin5 allele. Apart from the increase in mature fruit sugar and increases in some amino acids, metabolite profiling revealed few other metabolic perturbations in fruit from IL9-2-5. The only other major change was a dramatic increase in starch accumulation at earlier stages of fruit metabolism. This occurred without any increase in the activity of the enzymes of sucrose metabolism or starch synthesis and may therefore be driven by increased availability of sucrose. We conclude that the major factor that leads to increased fruit sugar in IL9-2-5 is an increase in the capacity to take up sucrose unloaded from the phloem.

Amino Acids↗

PubMed2005

Enhanced photosynthetic performance and growth as a consequence of decreasing mitochondrial malate dehydrogenase activity in transgenic tomato plants.

Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the mitochondrial malate dehydrogenase gene in the antisense orientation and exhibiting reduced activity of this isoform of malate dehydrogenase show enhanced photosynthetic activity and aerial growth under atmospheric conditions (360 ppm CO2). In comparison to wild-type plants, carbon dioxide assimilation rates and total plant dry matter were up to 11% and 19% enhanced in the transgenics, when assessed on a whole-plant basis. Accumulation of carbohydrates and redox-related compounds such as ascorbate was also markedly elevated in the transgenics. Also increased in the transgenic plants was the capacity to use L-galactono-lactone, the terminal precursor of ascorbate biosynthesis, as a respiratory substrate. Experiments in which ascorbate was fed to isolated leaf discs also resulted in increased rates of photosynthesis providing strong indication for an ascorbate-mediated link between the energy-generating processes of respiration and photosynthesis. This report thus shows that the repression of this mitochondrially localized enzyme improves both carbon assimilation and aerial growth in a crop species.

Ascorbic Acid↗

PubMed2005

Identification and characterisation of the alpha and beta subunits of succinyl CoA ligase of tomato.

Despite the central importance of the TCA cycle in plant metabolism not all of the genes encoding its constituent enzymes have been functionally identified. In yeast, the heterodimeric protein succinyl CoA ligase is encoded for by two single-copy genes. Here we report the isolation of two tomato cDNAs coding for alpha- and one coding for the beta-subunit of succinyl CoA ligase. These three cDNAs were used to complement the respective Saccharomyces cerevisiae mutants deficient in the alpha- and beta-subunit, demonstrating that they encode functionally active polypeptides. The genes encoding for the subunits were expressed in all tissues, but most strongly in floral and leaf tissues, with equivalent expression of the two alpha-subunit genes being expressed to equivalent levels in all tissues. In all instances GFP fusion expression studies confirmed an expected mitochondrial location of the proteins encoded. Following the development of a novel assay to measure succinyl CoA ligase activity, in the direction of succinate formation, the evaluation of the maximal catalytic activities of the enzyme in a range of tissues revealed that these paralleled those of mRNA levels. We also utilized this assay to perform a preliminary characterisation of the regulatory properties of the enzyme suggesting allosteric control of this enzyme which may regulate flux through the TCA cycle in a manner consistent with its position therein.

Amino Acid Sequence↗

PubMed2004

Deficiency of a plastidial adenylate kinase in Arabidopsis results in elevated photosynthetic amino acid biosynthesis and enhanced growth.

An Arabidopsis (Arabidopsis thaliana) L. Heynh mutant deficient in an isoform of adenylate kinase (ADK; At2g37250) was isolated by reverse genetics. It contains a T-DNA insertion 377 bp downstream of the start point of transcription. The mutant lacks At2g37250 transcripts and has a mild reduction in total cellular ADK activity. Green fluorescent protein-fusion based cellular localization experiments, carried out with the full-length At2g37250, suggested a plastidial localization for this isoform. In keeping with this observation, organelle isolation experiments revealed that the loss in ADK activity was confined to the inner plastid. This plastid stroma ADK gene was found to be expressed tissue constitutively but at much higher levels in illuminated leaves. Phenotypic and biochemical analyses of the mutant revealed that it exhibited higher amino acid biosynthetic activity in the light and was characterized by an enhanced root growth. When the mutant was subjected to either continuous light or continuous dark, growth phenotypes were also observed in the shoots. While the levels of adenylates were not much altered in the leaves, the pattern of change observed in the roots was consistent with the inhibition of an ATP-consuming reaction. Taken together, these data suggest a role for the plastid stromal ADK in the coordination of metabolism and growth, but imply that the exact importance of this isoform is tissue dependent.

Adenylate Kinase↗

PubMed2004

Zooming in on a quantitative trait for tomato yield using interspecific introgressions.

To explore natural biodiversity we developed and examined introgression lines (ILs) containing chromosome segments of wild species (Solanum pennellii) in the background of the cultivated tomato (S. lycopersicum). We identified Brix9-2-5, which is a S. pennellii quantitative trait locus (QTL) that increases sugar yield of tomatoes and was mapped within a flower- and fruit-specific invertase (LIN5). QTL analysis representing five different tomato species delimited the functional polymorphism of Brix9-2-5 to an amino acid near the catalytic site of the invertase crystal, affecting enzyme kinetics and fruit sink strength. These results underline the power of diverse ILs for high-resolution perspectives on complex phenotypes.

Alleles↗

PubMed2004

Expression of a yeast acetyl CoA hydrolase in the mitochondrion of tobacco plants inhibits growth and restricts photosynthesis.

Acetyl Coenzyme A (acetyl CoA) is required in the mitochondria to fuel the operation of the Krebs cycle and within the cytosolic, peroxisomal and plastidial compartments wherein it acts as the immediate precursor for a wide range of anabolic functions. Since this metabolite is impermeable to membranes it follows that discrete pathways both for its synthesis and for its utilization must be present in each of these organelles and that the size of the various compartmented pools are independently regulated. To determine the specific role of acetyl CoA in the mitochondria we exploited a transgenic approach to introduce a yeast acetyl CoA hydrolase (EC 3.1.2.1.) into this compartment in tobacco plants. Despite the facts that the introduced enzyme was correctly targeted and that there were marked reductions in the levels of citrate and malate and an increase in the acetate content of the transformants, the transgenic plants surprisingly exhibited increased acetyl CoA levels. The lines were further characterised by a severe growth retardation, abnormal leaf colouration and a dramatic reduction in photosynthetic activity correlated with a marked reduction in the levels of transcripts of photosynthesis and in the content of photosynthetic pigments. The altered rate of photosynthesis in the transgenics was also reflected by a modified carbon partitioning in leaves of these lines, however, further studies revealed that this was most likely caused by a decreased source to sink transport of carbohydrate. In summary these results suggest that the content of acetyl CoA is under tight control and that alterations in the level of this central metabolite have severe metabolic and developmental consequences in tobacco.

Acetate-CoA Ligase↗

PubMed2004

Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron transport.

The respiratory pathways of glycolysis, the tricarboxylic acid (TCA) cycle and the mitochondrial electron transport chain are ubiquitous throughout nature. They are essential for both energy provision in heterotrophic cells and a wide range of other physiological functions. Although the series of enzymes and proteins that participate in these pathways have long been known, their regulation and control are much less well understood. Further complexity arises due to the extensive interaction among these pathways in particular, and also between cytosolic and mitochondrial metabolism in general. These interactions include those between mitochondrial function in the photosynthetic and photorespiratory processes, amino-acid biosynthesis and the regulation of cellular redox. Recently, a wide range of molecular and biochemical strategies have been adopted to elucidate the functional significance of these interactions.

Biological Transport↗

PubMed2004

Heard it through the grapevine? ABA and sugar cross-talk: the ASR story.

Recently, Birsen Cakir et al. have defined a role for the abscisic acid signalling ASR peptide in the regulation of heterotrophic sugar metabolism, highlighting the importance of cross-talk between these signal transduction pathways in a broad species range. Analyses of developmental changes in tomato fruit metabolism suggest that the import of hexoses is also similarly regulated in this species. Taken together with the previously defined characteristics of ASP peptides, these findings should facilitate further dissection of the changing sugar and hormonal signal circuitry during fruit development.

Glucose↗

PubMed2003

Reduced expression of aconitase results in an enhanced rate of photosynthesis and marked shifts in carbon partitioning in illuminated leaves of wild species tomato.

Wild species tomato (Lycopersicon pennellii) plants bearing a genetic lesion in the gene encoding aconitase (Aco-1; aconitate hydratase EC 4.2.1.3) were characterized at molecular and biochemical levels. The genetic basis of this lesion was revealed by cloning the wild-type and mutant alleles. The mutation resulted in lowered expression of the Aco-1 transcript and lowered levels of both cytosolic and mitochondrial aconitase protein and activity. After in silico analysis, we concluded that in the absence of a recognizable target sequence, the best explanation for the dual location of this protein is inefficient targeting. Biochemical analysis of leaves of the Aco-1 accession suggested that they exhibited a restricted flux through the Krebs cycle and reduced levels of Krebs cycle intermediates but were characterized by elevated adenylate levels and an enhanced rate of CO2 assimilation. Furthermore, the analysis of both steady-state metabolite levels and metabolic fluxes revealed that this accession also exhibited elevated rates of photosynthetic Suc synthesis and a corresponding increase in fruit yield. Therefore, we conclude that the Krebs cycle normally competes with the Suc synthetic pathway for carbon but is not essential for the supply of energy to fuel the operation of this pathway.

Aconitate Hydratase↗