Strawberry (Fragaria × ananassa) fruits contain high concentrations of flavonoids. In unripe strawberries, the flavonoids are mainly represented by proanthocyanidins (PAs), while in ripe fruits the ...red-coloured anthocyanins also accumulate. Most of the structural genes leading to PA biosynthesis in strawberry have been characterized, but no information is available on their transcriptional regulation. In Arabidopsis thaliana the expression of the PA biosynthetic genes is specifically induced by a ternary protein complex, composed of AtTT2 (AtMYB123), AtTT8 (AtbHLH042) and AtTTG1 (WD40-repeat protein).
A strategy combining yeast-two-hybrid screening and agglomerative hierarchical clustering of transcriptomic and metabolomic data was undertaken to identify strawberry PA regulators.
Among the candidate genes isolated, four were similar to AtTT2, AtTT8 and AtTTG1 (FaMYB9/FaMYB11, FabHLH3 and FaTTG1, respectively) and two encode putative negative regulators (FaMYB5 and FabHLH3Δ). Interestingly, FaMYB9/FaMYB11, FabHLH3 and FaTTG1 were found to complement the tt2-1, tt8-3 and ttg1-1 transparent testa mutants, respectively. In addition, they interacted in yeast and activated the Arabidopsis BANYULS (anthocyanidin reductase) gene promoter when coexpressed in Physcomitrella patens protoplasts.
Taken together, these results demonstrated that FaMYB9/FaMYB11, FabHLH3 and FaTTG1 are the respective functional homologues of AtTT2, AtTT8 and AtTTG1, providing new tools for modifying PA content and strawberry fruit quality.
Environmental stress factors such as drought, elevated temperature, salinity and rising CO
2 affect plant growth and pose a growing threat to sustainable agriculture. This has become a hot issue due ...to concerns about the effects of climate change on plant resources, biodiversity and global food security. Plant adaptation to stress involves key changes in the ‘-omic’ architecture. Here, we present an overview of the physiological and molecular programs in stress adaptation focusing on how genes, proteins and metabolites change after individual and multiple environmental stresses. We address the role which ‘-omics’ research, coupled to systems biology approaches, can play in future research on plants seemingly unable to adapt as well as those which can tolerate climatic change.
β-Carotene is overproduced in the alga Dunaliella salina in response to high light intensities. We have studied the effects of a sudden light increase on carotenoid and fatty acid metabolism using a ...flat panel photobioreactor that was run in turbidostat mode to ensure a constant light regime throughout the experiments. Upon the shift to an increased light intensity, β-carotene production commenced immediately. The first 4 h after induction were marked by constant intracellular levels of β-carotene (2.2 g LCV⁻¹), which resulted from identical increases in the production rates of cell volume and β-carotene. Following this initial phase, β-carotene productivity continued to increase while the cell volume productivity dropped. As a result, the intracellular β-carotene concentration increased reaching a maximum of 17 g LCV⁻¹ after 2 days of light stress. Approximately 1 day before that, the maximum β-carotene productivity of 30 pg cell⁻¹ day⁻¹ (equivalent to 37 mg LRV⁻¹ day⁻¹) was obtained, which was about one order of magnitude larger than the average productivity reported for a commercial β-carotene production facility, indicating a vast potential for improvement. Furthermore, by studying the light-induced changes in both β-carotene and fatty acid metabolism, it appeared that carotenoid overproduction was associated with oil globule formation and a decrease in the degree of fatty acid unsaturation. Our results indicate that cellular β-carotene accumulation in D. salina correlates with accumulation of specific fatty acid species (C16:0 and C18:1) rather than with total fatty acid content. Biotechnol. Bioeng. 2010;106: 638-648.
The taxonomic classification of the genus Vernonia Schreb is complex and, as yet, unclear. We here report the use of untargeted metabolomics approaches, followed by multivariate analyses methods and ...a phytochemical characterization of ten Vernonia species. Metabolic fingerprints were obtained by accurate mass measurements and used to determine the phytochemical similarities and differences between species through multivariate analyses approaches. Principal component analysis based on the relative levels of 528 metabolites, indicated that the ten species could be clustered into four groups. Thereby, V. polyanthes was the only species with presence of flavones chrysoeriol-7-O-glycuronyl, acacetin-7-O-glycuronyl and sesquiterpenes lactones piptocarphin A and piptocarphin B, while glaucolide A was detected in both V. brasiliana and V. polyanthes, separating these species from the two other species of the Vernonanthura group. Species from the Lessingianthus group were unique in showing a positive response in the foam test, suggesting the presence of saponins, which could be confirmed by metabolite annotation. V. rufogrisea showed a great variety of sesquiterpene lactones, placing this species into a separate group. Species within the Chrysolaena group were unique in accumulating clovamide. Our results of LC-MS-based profiling combined with multivariate analyses suggest that metabolomics approaches, such as untargeted LC-MS, may be potentially used as a large-scale chemotaxonomical tool, in addition to classical morphological and cytotaxonomical approaches, in order to facilitate taxonomical classifications.
For the description of the metabolome of an organism, the development of common metabolite databases is of utmost importance. Here we present the Metabolome Tomato Database (MoTo DB), a metabolite ...database dedicated to liquid chromatography-mass spectrometry (LC-MS)- based metabolomics of tomato fruit (Solanum lycopersicum). A reproducible analytical approach consisting of reversed-phase LC coupled to quadrupole time-of-flight MS and photodiode array detection (PDA) was developed for large-scale detection and identification of mainly semipolar metabolites in plants and for the incorporation of the tomato fruit metabolite data into the MoTo DB. Chromatograms were processed using software tools for mass signal extraction and alignment, and intensity-dependent accurate mass calculation. The detected masses were assigned by matching their accurate mass signals with tomato compounds reported in literature and complemented, as much as possible, by PDA and MS/MS information, as well as by using reference compounds. Several novel compounds not previously reported for tomato fruit were identified in this manner and added to the database. The MoTo DB is available at http://appliedbioinformatics.wur.nl and contains all information so far assembled using this LC-PDA-quadrupole time-of-flight MS platform, including retention times, calculated accurate masses, PDA spectra, MS/MS fragments, and literature references. Unbiased metabolic profiling and comparison of peel and flesh tissues from tomato fruits validated the applicability of the MoTo DB, revealing that all flavonoids and α-tomatine were specifically present in the peel, while several other alkaloids and some particular phenylpropanoids were mainly present in the flesh tissue.
The cuticle covering plants' aerial surfaces is a unique structure that plays a key role in organ development and protection against diverse stress conditions. A detailed analysis of the tomato ...colorless-peel y mutant was carried out in the framework of studying the outer surface of reproductive organs. The y mutant peel lacks the yellow flavonoid pigment naringenin chalcone, which has been suggested to influence the characteristics and function of the cuticular layer. Large-scale metabolic and transcript profiling revealed broad effects on both primary and secondary metabolism, related mostly to the biosynthesis of phenylpropanoids, particularly flavonoids. These were not restricted to the fruit or to a specific stage of its development and indicated that the y mutant phenotype is due to a mutation in a regulatory gene. Indeed, expression analyses specified three R2R3-MYB-type transcription factors that were significantly down-regulated in the y mutant fruit peel. One of these, SlMYB12, was mapped to the genomic region on tomato chromosome 1 previously shown to harbor the y mutation. Identification of an additional mutant allele that co-segregates with the colorless-peel trait, specific down-regulation of SlMYB12 and rescue of the y phenotype by overexpression of SlMYB12 on the mutant background, confirmed that a lesion in this regulator underlies the y phenotype. Hence, this work provides novel insight to the study of fleshy fruit cuticular structure and paves the way for the elucidation of the regulatory network that controls flavonoid accumulation in tomato fruit cuticle.
In dietary polyphenol exposure studies, annotation and identification of urinary metabolites present at low (micromolar) concentrations are major obstacles. To determine the biological activity of ...specific components, it is necessary to have the correct structures and the quantification of the polyphenol-derived conjugates present in the human body. We present a procedure for identification and quantification of metabolites and conjugates excreted in human urine after single bolus intake of black or green tea. A combination of a solid-phase extraction (SPE) preparation step and two high pressure liquid chromatography (HPLC)-based analytical platforms was used, namely, accurate mass fragmentation (HPLC-FTMSn) and mass-guided SPE-trapping of selected compounds for nuclear magnetic resonance spectroscopy (NMR) measurements (HPLC-TOFMS-SPE-NMR). HPLC-FTMSn analysis led to the annotation of 138 urinary metabolites, including 48 valerolactone and valeric acid conjugates. By combining the results from MSn fragmentation with the one-dimensional (1D)-1H NMR spectra of HPLC-TOFMS-SPE-trapped compounds, we elucidated the structures of 36 phenolic conjugates, including the glucuronides of 3′,4′-di- and 3′,4′,5′-trihydroxyphenyl-γ-valerolactone, three urolithin glucuronides, and indole-3-acetic acid glucuronide. We also obtained 26 h-quantitative excretion profiles for specific valerolactone conjugates. The combination of the HPLC-FTMSn and HPLC-TOFMS-SPE-NMR platforms results in the efficient identification and quantification of less abundant phenolic conjugates down to nanomoles of trapped amounts of metabolite corresponding to micromolar metabolite concentrations in urine.
To take full advantage of the power of functional genomics technologies and in particular those for metabolomics, both the analytical approach and the strategy chosen for data analysis need to be as ...unbiased and comprehensive as possible. Existing approaches to analyze metabolomic data still do not allow a fast and unbiased comparative analysis of the metabolic composition of the hundreds of genotypes that are often the target of modern investigations. We have now developed a novel strategy to analyze such metabolomic data. This approach consists of (1) full mass spectral alignment of gas chromatography (GC)-mass spectrometry (MS) metabolic profiles using the MetAlign software package, (2) followed by multivariate comparative analysis of metabolic phenotypes at the level of individual molecular fragments, and (3) multivariate mass spectral reconstruction, a method allowing metabolite discrimination, recognition, and identification. This approach has allowed a fast and unbiased comparative multivariate analysis of the volatile metabolite composition of ripe fruits of 94 tomato (Lycopersicon esculentum Mill.) genotypes, based on intensity patterns of >20,000 individual molecular fragments throughout 198 GC-MS datasets. Variation in metabolite composition, both between- and within-fruit types, was found and the discriminative metabolites were revealed. In the entire genotype set, a total of 322 different compounds could be distinguished using multivariate mass spectral reconstruction. A hierarchical cluster analysis of these metabolites resulted in clustering of structurally related metabolites derived from the same biochemical precursors. The approach chosen will further enhance the comprehensiveness of GC-MS-based metabolomics approaches and will therefore prove a useful addition to nontargeted functional genomics research.
α-tomatineis an antifungal glycoalkaloid that provides basal defense to tomato (Solanum lycopersicum). However, tomato pathogens overcome this basal defense barrier by the secretion of tomatinases ...that degrade α-tomatineinto the less fungitoxic compounds β-tomatine and tomatidine. Although pathogenic on tomato, it has been reported that the biotrophic fungus Cladosporium fulvum is unable to detoxify α-tomatine.
Here, we present a functional analysis of the glycosyl hydrolase (GH10), CfTom1, which is orthologous to fungal tomatinases.
We show that C. fulvum hydrolyzes α-tomatineinto tomatidine in vitro and during the infection of tomato, which is fully attributed to the activity of CfTom1, as shown by the heterologous expression of this enzyme in tomato. Accordingly, Δcftom1 mutants of C. fulvum are more sensitive to α-tomatineand are less virulent than the wild-type fungus on tomato. Although α-tomatineis thought to be localized in the vacuole, we show that it is also present in the apoplast, where it is hydrolyzed by CfTom1 on infection. The accumulation of tomatidine during infection appears to be toxic to tomato cells and does not suppress defense responses, as suggested previously.
Altogether, our results show that CfTom1 is responsible for the detoxification of α-tomatine by C. fulvum, and is required for full virulence of this fungus on tomato.
The influence of internal (genetic and developmental) and external (environmental) factors on levels of flavonoid gene transcripts, enzyme activity and metabolites was studied in fruit of six ...cultivated strawberry (Fragaria x ananassa Duch.) genotypes grown at two Italian locations. Gene expression and enzyme activity showed development- and genotype-associated patterns, revealing gene coordination. Analysis clarified the regulation mechanism of the hydroxylation status of the B-ring of the major flavonoid pools and pointed out examples of genotype-specific post-transcriptional regulation mechanisms and key steps of pathway regulation in strawberry fruits. Metabolite profiles were strongly affected by development and genotype. Flavan-3-ols, their proanthocyanidin (PA) derivatives and anthocyanins were the most abundant metabolites. Flavonol levels and PA-associated traits (epicatechin/catechin ratio and mean degree of polymerization) showed significant environmental effects. Multivariate and correlation analyses determined the relationships among genes, enzymes and metabolites. The combined molecular and biochemical information elucidated more in depth the role of genetic and environmental factors on flavonoid metabolism during strawberry fruit development, highlighting the major impact of developmental processes, and revealing genotype-dependent differences and environmental effects on PA-related traits.
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