Biological and Mathematical Models for Glutamate Metabolism in Tomatoes - Term Paper Example

BIOLOGICAL AND MATHEMATICAL MODELS FOR GLUTAMATE METABOLISM IN TOMATOES Abstracts Fruit or vegetable? - Botanically, a tomato is the ovary, together with its seeds, of a flowering plant. Lycopene, one of nature's most powerful antioxidants, is present in tomatoes, and especially when tomatoes are cooked, it has been found to benefit the heart among other things. We begin with a brief description of evolutionary features of glutamate in the world of molecular biology, it properties and functions, and its role in the metabolism of tomatoes, biochemical approaches and the analysis of intermediates in naturally occurring mutants, especially the tomato. The properties of glutamate and its model and structure gives us a reasonable knowledge on the metabolism of the glutamate in the ripening process of the fleshy fruits, especially the tomatoes and the reactive features of glutamate that account for its determinant role in metabolism and possibly its abundance in nature. INTRODUCTION 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. PROPERTIES OF GLUTAMATE The abundance of glutamate might be examined by considering the features and patterns of reactivity of the glutamate molecule; this can be approached by examining four of the contexts in which the properties of glutamate itself have proven consequential. The exercise might also be worthwhile because these properties were underrepresented at the earlier international symposium on glutamate (Filer et al. 1979 ). We begin with some comments on the overall structural motif in which the "entire molecule," so to speak, participates in its unique function, and then we will consider the biochemical/chemical reactivity of specific positions within the glutamate molecule. GLUTAMATE METABOLISM 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. In the consideration of 15N flux via the glutamine synthetase (GS) - glutamate synthase (GOGAT) cycle. The GS/GOGAT cycle is thought to be the primary pathway of ammonia assimilation in higher plants. In this cycle, 15NH3 is first incorporated into the amide-group of glutamine (Gln) in the reaction catalyzed by the chloroplast isoform of GS. The amide-group of Gln is then transferred to 2-oxoglutarate in the reaction catalyzed by the chloroplast-localized, ferredoxin-dependent GOGAT, producing 2 molecules of glutamate (Glu). One of these Glu molecules is randomly re-utilized in the synthesis of Gln [in this case transferring 15N label to the amino-group of Gln], and the second Glu is used in other reactions in the chloroplast, or exported to the cytosol for net amino acid synthesis In the greatly simplified model shown above, we have allowed exactly one-half of the Glu formed in the GOGAT reaction in the chloroplast to be exported to the cytoplasm [i.e. no utilization of either Glu or Gln in other reactions is envisaged to occur within the chloroplast]. Thus, the rate of Glu export from the chloroplast (r1) is the same as the rate of ammonia assimilation in the chloroplast (r1). The Glu that is exported to the cytosol is envisaged to be used in several reactions. In part, this Glu is used to sustain the synthesis of Gln by a second cytosolic isoform of GS (r2) [in this particular scenario, the flux via the cytosolic Gln pool corresponds to 10% of the total ammonia assimilation rate]. Note that the cytosolic Gln pool receives 15N in the amide position from 15NH3, and in the amino position from the cytosolic Glu pool. The cytosolic Glu is, in part, envisaged to be used in the synthesis of two amino acids; proline (Pro) and gamma-aminobutyrate (GABA). In the case of Pro synthesis (r10), the pools of intermediates (gamma-glutamylphosphate, glutamic semialdehyde and pyrroline-5-carboxylate) are envisaged to be negligible. GABA synthesis is envisaged to occur in a single step (r13); decarboxylation of Glu. Both GABA and Pro can be metabolized back to Glu; in the former case by transamination (r14), and in the latter case by oxidation (r9). In this greatly simplified model we have not considered the potential role of the mitochondrion in processes such as proline oxidation. The cytosolic Gln, Glu and Pro pools are envisaged to be utilized in the synthesis of protein in the cytosol, at rates r5, r6 and r11, respectively [note that since GABA is a non-protein amino acid, a similar fate for GABA is not considered]. Small portions of the newly synthesized cytosolic Gln, Glu and Pro pools are also envisaged to be sequestered in the vacuole, at rates r4, r7 and r12. For simplicity, a vacuolar pool of GABA is not considered. "Other" unspecified metabolic fates of Gln, Glu and GABA make up the balance of the fluxes (e.g. for Gln "other" (r3) could include synthesis of asparagine, histidine and tryptophan; for Glu "other" (r8) could include transamination to aspartate, glycine, alanine, and many other amino acids; for GABA "other" (r15) could include transamination to alanine). Note that for each of the pools (except the vacuolar and protein pools) the total influx to each pool is equal to the total efflux from the corresponding pool (e.g. r2 = r3 + r4 + r5; r10 = r9 + r11 + r12; r1 + r14 + r9 = r2+ r13 + r10 + r6 + r7 + r8). Thus, the chloroplastic and cytosolic pools of the amino acids remain constant, while the vacuolar and protein-bound pools expand with time. The average isotope abundances and total pool sizes of the total free pools have been plotted; for example, the isotope abundance for Gln-amide corresponds to the average isotope abundance of the chloroplastic, cytosolic and vacuolar Gln-amide pools. Because the Gln-amide and Gln-amino nitrogen pool sizes are of exactly the same size, only the Gln-amino nitrogen pool is shown. When different option buttons are checked, this permits simulation of the individual isotope abundances and pool sizes of the chloroplastic (Chl), cytosolic (Cyt), vacuolar (Vac), and protein-bound (Prot.) pools, using identical flux and pool size assumptions. It is only the chloroplastic pools which reveal the expected order of labeling of intermediates for the operation of the GS/GOGAT cycle; i.e. Gln-amide > Glu > Gln-amino. The presence of a second, more slowly-turning over Gln pool in the cytosol, and a large vacuolar pool of Gln, substantially distorts the labeling patterns for the total free pool of Gln, such that Glu rather than Gln-amide, is the most heavily labeled product during the later part of the "pulse" phase of the labeling time-course 1 This work was supported by the Max Planck Society (in the form of a Max-Planck partner laboratory grant to F.C. and A.R.F.) and two independent grants in the BMBF GABI Program (to B.U. and to A.R.F. and M.-I.Z.), as well as by the Biotechnology and Biological Sciences Research Council (to C.B. and L.J.S.), CONICET, INTA, and EMBO (to F.C.). Role for glutamate decarboxylase during tomato ripening Edible coatings could be effective tools for delaying the ripening process of fruits. Alginate or zein as edible coatings were assayed in tomato in order to maintain parameters related to quality during postharvest storage. Coated tomatoes showed lower respiration rate and ethylene production than control ones, with a twofold lower concentration of ethylene precursor. In addition, the evolution of parameters related to tomato quality losses, such as softening, colour evolution and weight loss, was significantly delayed (4-6 days on average) in coated tomatoes as compared to controls. Thereafter, sugars, organic acids (and especially ascorbic acid) and scores from sensory analysis remained at much higher levels at the end of storage in treated than in control tomatoes. Coatings based on alginate or zein could be effective tools for delaying the tomato-ripening process during postharvest storage, and in turn maintaining tomato quality and its acceptability by consumers The ripening characteristics of modified tomatoes (Lycopersicon esculentum Mill cv Ailsa Craig), which express antisense RNA to polygalacturonase (PG) and thus have very low activity of this enzyme, were compared with control fruit. Previous studies of these fruits showed that although PG activity was reduced to approximately 1% of that in untransformed tomatoes, this reduction had no effect on softening. Further detailed mechanical assessments have now been performed which revealed small, but significant differences in the fruit ripening characteristics between control and antisense fruits. Compression along the polar axis of the PG antisense fruit was significantly reduced relative to the control. Deformation at failure (h), modulus (M), coefficient of compression (Kc), and coefficient of shear (Ks) values from probe tests along the equatorial axis also indicated that the antisense fruit was firmer. Although these significant differences in texture properties were observed between the antisense fruit and the control, they were quite limited in extent, as compared to the normal evolution observed during ripening. Scanning electron microscope studies on the sub-exocarpic region of the pericarp showed that cell wall separation was reduced in the ripe antisense tomatoes, and this may explain the change in mechanical properties. Optical measurements made with both Hunter Color Difference (Hunter Lab Ltd, Fairfax. VA, USA) and Micromatch 2000 (ICS Texicon Ltd. Altrincham, UK) spectrophotometer systems showed that the a (green/red) component of colour of the antisense tomatoes was increased relative to that of the control samples. The effect of PG on softening and of cell wall breakdown on mechanical and optical properties is also considered. Antisense regulation, glutamate decarboxylase, Lycopersicon esculentum, ripening fruits. Glutamate is one of the a -amino acids that is synthesized during the first step of nitrogen metabolism in higher plants. Glutamine and asparagine, formed from glutamate, are distributed to plant tissues via the phloem and used for the synthesis of other amino acids and proteins. In higher plants, glutamate is present at high concentrations in phloem and it is found at high concentrations in the edible parts of plants. For example, tomato fruits contain 2,800 nmol g_1 fresh weight glutamate (17.2% of total free amino acids in cv. Platense; Boggio et al. 2000). Reports on the biosynthesis of amino acids in transgenic plants include, a report that the level of free lysine was increased 200-fold by the introduction, into tobacco, of a gene for dihydropicolinate synthase from E. coli (Glassman et al. 1988); a report that the level of free lysine was increased by introduction, into corn, of a gene for aspartate kinase from E. coli (Falco 1993); a report that the level of asparagine was increased 100-fold in seeds by introduction, into tobacco, of a gene for asparagine synthetase from pea (Brears et al. 1993); and a report that the level of tryptophan was increased 90-fold by introduction, into rice, of a gene for anthranilate synthetase from corn (Anderson et al. 1997). However, it is not easy to engineer high concentrations of glutamate in plants because glutamate provides the amino group for the biosynthesis of several amino acids, namely, asparagine, alanine, glycine, serine, proline and g -aminobutyric acid (GABA), and is also metabolized in various biosynthetic pathways. It has not yet proved possible, to our knowledge, to increase levels of glutamate in edible parts of plants by cross breeding. It was reported that, when the gene for glutamate dehydrogenase (GDH) from E. coli was introduced into tobacco and corn to engineer resistance to the herbicide phosphinothricin, the level of glutamate in roots was increased only 1.3- to 1.4-fold, as compared to control plants (Lightfoot and Long 1999). The glutamate content of fruits of transgenic tomato that harbored a gene for NADP-dependent GDH from Aspergillus nidulans was approximately twice that of wild-type fruits (Kisaka and Kida 2003). It is well known that GABA accumulates in storage organs, such as tomato fruits and sugar beet roots. It is also known that the accumulation of GABA is induced by environmental stresses, such as low temperature and heat shock (Streeter and Thompson, 1972; Reggiani et al. 1988; Menegus et al. 1989; Aurisano et al. 1995; Tomato plants were transformed with a plasmid that contained a the gene for glutamate decarboxylase (GAD) from Lycopersicon esculentum L. coupled, in the antisense orientation, with the constitutively active 35S promoter from cauliflower mosaic virus. Four independent transformants were obtained. In the fruits of these transgenic plants, the level of expression of GAD mRNA was lower than that in non-transgenic plants. When tomatoes were harvested six weeks after the first flowering, we found that the levels of total free amino acids in transgenic fruits were 1.2 to 3.2 times higher than those in non-transgenic plants. 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