Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts.

BACKGROUND: Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts. RESULTS: A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way. CONCLUSIONS: We have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data.

Stress and developmental responses of terpenoid biosynthetic genes in Cistus creticus subsp. creticus.

Plants, and specially species adapted in non-friendly environments, produce secondary metabolites that help them to cope with biotic or abiotic stresses. These metabolites could be of great pharmaceutical interest because several of those show cytotoxic, antibacterial or antioxidant activities. Leaves' trichomes of Cistus creticus ssp. creticus, a Mediterranean xerophytic shrub, excrete a resin rich in several labdane-type diterpenes with verified in vitro and in vivo cytotoxic and cytostatic activity against human cancer cell lines. Bearing in mind the properties and possible future exploitation of these natural products, it seemed interesting to study their biosynthesis and its regulation, initially at the molecular level. For this purpose, genes encoding enzymes participating in the early steps of the terpenoids biosynthetic pathways were isolated and their gene expression patterns were investigated in different organs and in response to various stresses and defence signals. The genes studied were the CcHMGR from the mevalonate pathway, CcDXS and CcDXR from the methylerythritol 4-phosphate pathway and the two geranylgeranyl diphosphate synthases (CcGGDPS1 and 2) previously characterized from this species. The present work indicates that the leaf trichomes are very active biosynthetically as far as it concerns terpenoids biosynthesis, and the terpenoid production from this tissue seems to be transcriptionally regulated. Moreover, the CcHMGR and CcDXS genes (the rate-limiting steps of the isoprenoids' pathways) showed an increase during mechanical wounding and application of defence signals (like meJA and SA), which is possible to reflect an increased need of the plant tissues for the corresponding metabolites.

Over-expression of a tomato N-acetyl-L-glutamate synthase gene (SlNAGS1) in Arabidopsis thaliana results in high ornithine levels and increased tolerance in salt and drought stresses.

A single copy of the N-acetyl-L-glutamate synthase gene (SlNAGS1) has been isolated from tomato. The deduced amino acid sequence consists of 604 amino acids and shows a high level of similarity to the predicted Arabidopsis NAGS1 and NAGS2 proteins. Furthermore, the N-terminus ArgB domain and the C-terminus ArgA domain found in SlNAGS1 are similar to the structural arrangements that have been reported for other predicted NAGS proteins. SlNAGS1 was expressed at high levels in all aerial organs, and at basic levels in seeds, whereas it was not detected at all in roots. SlNAGS1 transcript accumulation was noticed transiently in tomato fruit at the red-fruit stage. In addition, an increase of SlNAGS1 transcripts was detected in mature green tomato fruit within the first hour of exposure to low oxygen concentrations. Transgenic Arabidopsis plants have been generated expressing the SlNAGS1 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Three homozygous transgenic lines expressing the transgene (lines 1-7, 3-8, and 6-5) were evaluated further. All three transgenic lines showed a significant accumulation of ornithine in the leaves with line 3-8 exhibiting the highest concentration. The same lines demonstrated higher germination ability compared to wild-type (WT) plants when subjected to 250 mM NaCl. Similarly, mature plants of all three transgenic lines displayed a higher tolerance to salt and drought stress compared to WT plants. Under most experimental conditions, transgenic line 3-8 performed best, while the responses obtained from lines 1-7 and 6-5 depended on the applied stimulus. To our knowledge, this is the first plant NAGS gene to be isolated, characterized, and genetically modified.

Expression profiling of ascorbic acid-related genes during tomato fruit development and ripening and in response to stress conditions.

L-ascorbate (the reduced form of vitamin C) participates in diverse biological processes including pathogen defence mechanisms, and the modulation of plant growth and morphology, and also acts as an enzyme cofactor and redox status indicator. One of its chief biological functions is as an antioxidant. L-ascorbate intake has been implicated in the prevention/alleviation of varied human ailments and diseases including cancer. To study the regulation of accumulation of this important nutraceutical in fruit, the expression of 24 tomato (Solanum lycopersicon) genes involved in the biosynthesis, oxidation, and recycling of L-ascorbate during the development and ripening of fruit have been characterized. Taken together with L-ascorbate abundance data, the results show distinct changes in the expression profiles for these genes, implicating them in nodal regulatory roles during the process of L-ascorbate accumulation in tomato fruit. The expression of these genes was further studied in the context of abiotic and post-harvest stress, including the effects of heat, cold, wounding, oxygen supply, and ethylene. Important aspects of the hypoxic and post-anoxic response in tomato fruit are discussed. The data suggest that L-galactose-1-phosphate phosphatase could play an important role in regulating ascorbic acid accumulation during tomato fruit development and ripening.

Physiological and molecular responses of the isoprenoid biosynthetic pathway in a drought-resistant Mediterranean shrub, Cistus creticus exposed to water deficit.

The goal of the present research was to obtain new insights into the mechanisms underlying drought stress resistance in plants. Specifically, we evaluated changes in the expression of genes encoding enzymes involved in isoprenoid biosynthesis, together with the levels of the corresponding metabolites (chlorophylls, carotenoids, tocopherols and abscisic acid), in a drought-resistant Mediterranean shrub, Cistus creticus grown under Mediterranean field conditions. Summer drought led to reductions in the relative leaf water content (RWC) by 25%, but did not alter the maximum efficiency of PSII, indicating the absence of damage to the photosynthetic apparatus. While the expression of genes encoding C. creticus chlorophyll a oxygenase/chlorophyll b synthase (CAO) and phytoene synthase (PSY) were not affected by water deficit, the genes encoding homogentisate phytyl-transferase (HPT) and 9-cis-epoxycarotenoid dioxygenase (NCED) were induced in water-stressed (WS) plants. Drought-induced changes in gene expression were observed at early stages of drought and were strongly correlated with levels of the corresponding metabolites, with simultaneous increases in abscisic acid and alpha-tocopherol levels of up to 4-fold and 62%, respectively. Furthermore, alpha-tocopherol levels were strongly positively correlated with abscisic acid contents, but not with the levels of jasmonic acid and salicylic acid. We conclude that the abscisic acid and tocopherol biosynthetic pathway may be regulated at the transcript level in WS C. creticus plants, and that the genes encoding HPT and NCED may play a key role in the drought stress resistance of this Mediterranean shrub by modulating abscisic acid and tocopherol biosynthesis.

Transcriptome analysis approaches for the isolation of trichome-specific genes from the medicinal plant Cistus creticus subsp. creticus.

Cistus creticus subsp. creticus is a plant of intrinsic scientific interest due to the distinctive pharmaceutical properties of its resin. Labdane-type diterpenes, the main constituents of the resin, exhibit considerable antibacterial and cytotoxic activities. In this study chemical analysis of isolated trichomes from different developmental stages revealed that young leaves of 1-2 cm length displayed the highest content of labdane-type diterpenes (80 mg/g fresh weight) whereas trichomes from older leaves (2-3 or 3-4 cm) exhibited gradual decreased concentrations. A cDNA library was constructed enriched in transcripts from trichomes isolated from young leaves, which are characterized by high levels of labdane-type diterpenes. Functional annotation of 2,022 expressed sequence tags (ESTs) from the trichome cDNA library based on homology to A. thaliana genes suggested that 8% of the putative identified sequences were secondary metabolism-related and involved primarily in flavonoid and terpenoid biosynthesis. A significant proportion of the ESTs (38%) displayed no significant similarity to any other DNA deposited in databases, indicating a yet unknown function. Custom DNA microarrays constructed with 1,248 individual clones from the cDNA library facilitated transcriptome comparisons between trichomes and trichome-free tissues. In addition, gene expression studies in various Cistus tissues and organs for one of the genes highlighted as the most differentially expressed by the microarray experiments revealed a putative sesquiterpene synthase with a trichome-specific expression pattern. Full length cDNA isolation and heterologous expression in E. coli followed by biochemical analysis, led to the characterization of the produced protein as germacrene B synthase.

Isolation and functional analysis of two Cistus creticus cDNAs encoding geranylgeranyl diphosphate synthase.

Cistus creticus ssp. creticus is an indigenous shrub of the Mediterranean area. The glandular trichomes covering its leaf surfaces secrete a resin called "ladanum", which among others contains a number of specific labdane-type diterpenes that exhibit antibacterial and antifungal action as well as in vitro and in vivo cytotoxic and cytostatic activity against human cancer cell lines. In view of the properties and possible future exploitation of these metabolites, it was deemed necessary to study the geranylgeranyl diphosphate synthase enzyme (GGDPS, EC 2.5.1.30), a short chain prenyltransferase responsible for the synthesis of the precursor molecule of all diterpenes. In this work, we present the cloning, functional characterisation and expression profile at the gene and protein levels of two differentially expressed C. creticus full-length cDNAs, CcGGDPS1 and CcGGDPS2. Heterologous yeast cell expression system showed that these cDNAs exhibited GGDPS enzyme activity. Gene and protein expression analyses suggest that this enzyme is developmentally and tissue-regulated showing maximum expression in trichomes and smallest leaves (0.5-1.0cm). This work is the first attempt to study the terpenoid biosynthesis at the molecular level in C. creticus ssp. creticus.

Characterisation of the gene family encoding acetoacetyl-CoA thiolase in Arabidopsis.

Thiolases are ubiquitous enzymes involved in many essential biochemical processes. Biosynthetic thiolases, also known as acetoacetyl-CoA thiolases (AACT), catalyse a reversible Claisen-type condensation of two acetyl-CoA molecules to form acetoacetyl-CoA. Here, we report the characterisation of two genes from Arabidopsis thaliana L., ACT1 and ACT2, which encode two closely related AACT isoforms (AACT1 and AACT2, respectively). Transient expression of constructs encoding AACT1 and AACT2 fused to GFP revealed that the two proteins show a different subcellular localisation. While AACT1 is found in peroxisomes, AACT2 localises in the cytosol and the nucleus. The peroxisomal localisation of AACT1 depends on the presence of a C-terminal peroxisomal targeting sequence (PTS1) motif (Ser-Ala-Leu) not previously found in other organisms. ACT1 and ACT2 genes are also differentially expressed. Whereas ACT2 is expressed at relatively high level in all plant tissues, the expression of ACT1 is restricted to roots and inflorescences and its transcript is present at very low levels. The obtained results are in agreement with the involvement of AACT2 in catalysing the first step of the mevalonate pathway. The metabolic function of AACT1 is not clear at present, although its particular peroxisomal localisation might exclude a role in isoprenoid biosynthesis.

Identification and expression profiling of low oxygen regulated genes from Citrus flavedo tissues using RT-PCR differential display.

The molecular basis for the adaptation of fruit tissues to low oxygen treatments remains largely unknown. RT-PCR differential display (DD) was employed to isolate anoxic and/or hypoxic genes whose expression responded to short, low-oxygen regimes. This approach led to the isolation, cloning, successful sequencing, and bioinformatic analysis of 98 transcripts from Citrus flavedo tissues that were differentially expressed in DD gels in response to 0, 0.5, 3, and 21% O(2) for 24 h. RNA blot analysis of 25 DD clones revealed that 11 genes were induced under hypoxia and/or anoxia, 11 exhibited constitutive expression and three transcripts were suppressed by low oxygen levels. Almost half of the DD cDNAs were either of unknown function or shared no apparent homology to any expressed sequences in the GenBank/EMBL databases. Six DD genes were similar to molecules of the following functions: C-compound and carbohydrate utilization, plant development, amino acid metabolism, and biosynthesis of brasinosteroids. Time-course and stress-related experiments of low O(2)-regulated genes indicated that these genes responded differently in terms of their earliness, band intensity, and their specificity to stresses, showing that some of them can be termed hypoxia- or anoxia-induced genes.

Differential expression of the ascorbate oxidase multigene family during fruit development and in response to stress.

Ascorbate oxidase (AO, EC 1.10.3.3) is a member of the multicopper oxidases family. It catalyzes the oxidation of ascorbic acid (AA) to dehydroascorbic acid (DHA) via monodehydroascorbate (MDHA), with the concomitant reduction of molecular oxygen to water. In melon (Cucumis melo), ascorbate oxidase is encoded by a multigene family comprising at least four genes. Here, we present the detailed characterization of two melon AO genes, CmAO1 and CmAO4. Gene-specific expression studies of the AO gene family in melon revealed that only CmAO1 and CmAO4 are transcriptionally active and differentially regulated dependent on tissue, developmental stage and external stimuli. Transcripts of the CmAO1 gene are present in floral and fruit tissues, whereas CmAO4 mRNA preferentially accumulates in vegetative tissues. CmAO genes were not detected in melon seeds, but CmAO4 expression is activated upon germination. CmAO4 mRNA steady-state levels are also regulated in response to wounding and heat stress, by hormones (abscisic acid, salicylic acid and jasmonates), AA and copper. These findings suggest that AO gene expression is transcriptionally regulated during fruit development and in response to hormonal cues associated with the control of cell growth and the stress response.

Molecular characterization and expression studies during melon fruit development and ripening of L-galactono-1,4-lactone dehydrogenase.

The last step of ascorbic acid (AA) biosynthesis is catalysed by the enzyme L-galactono-1,4-lactone dehydrogenase (GalLDH, EC 1.3.2.3), located on the inner mitochondrial membrane. The enzyme converts L-galactono-1,4-lactone to ascorbic acid (AA). In this work, the cloning and characterization of a GalLDH full-length cDNA from melon (Cucumis melo L.) are described. Melon genomic DNA Southern analysis indicated that CmGalLDH was encoded by a single gene. CmGalLDH mRNA accumulation was detected in all tissues studied, but differentially expressed during fruit development and seed germination. It is hypothesized that induction of CmGalLDH gene expression in ripening melon fruit contributes to parallel increases in the AA content and so playing a role in the oxidative ripening process. Higher CmGalLDH message abundance in light-grown seedlings compared with those raised in the dark suggests that CmGalLDH expression is regulated by light. Finally, various stresses and growth regulators resulted in no significant change in steady state levels of CmGalLDH mRNA in 20-d-old melon seedlings. To the authors' knowledge, this is the first report of GalLDH transcript induction in seed germination and differential gene expression during fruit ripening.

Over-expression of ascorbate oxidase in the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox states and increased sensitivity to ozone.

Transgenic tobacco ( Nicotiana tabacum L. cv. Xanthi) plants expressing cucumber ascorbate oxidase (EC.1.10.3.3) were used to examine the role of extracellular ascorbic acid in mediating tolerance to the ubiquitous air pollutant, ozone (O(3)). Three homozygous transgenic lines, chosen on the basis of a preliminary screen of AO activity in the leaves of 29 lines, revealed up to a 380-fold increase in AO activity, with expression predominantly associated with leaf cell walls. Over-expression of AO resulted in no change in the total ascorbate content recovered in apoplast washing fluid, but the redox state of ascorbate was reduced from 30% in wild-type leaves to below the threshold for detection in transgenic plants. Levels of ascorbic acid and glutathione in the symplast were not affected by AO over-expression, but the redox state of ascorbate was reduced, while that of glutathione was increased. AO over-expressing plants exposed to 100 nmol mol(-1) ozone for 7 h day(-1) exhibited a substantial increase in foliar injury, and a greater pollutant-induced reduction in both the light-saturated rate of CO(2) assimilation and the maximum in vivo rate of ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation, compared with wild-type plants. Transgenic plants also exhibited a greater decline in CO(2) assimilation rate when exposed to a brief ozone episode (300 nmol mol(-1) for 8 h). Stomatal conductance, hence O(3) uptake, was unaffected by AO over-expression. Our findings illustrate the important role played by ascorbate redox state and sub-cellular compartmentation in mediating the tolerance of plants to ozone-induced oxidative stress.