The genetics of plant metabolism.

Variation for metabolite composition and content is often observed in plants. However, it is poorly understood to what extent this variation has a genetic basis. Here, we describe the genetic analysis of natural variation in the metabolite composition in Arabidopsis thaliana. Instead of focusing on specific metabolites, we have applied empirical untargeted metabolomics using liquid chromatography-time of flight mass spectrometry (LC-QTOF MS). This uncovered many qualitative and quantitative differences in metabolite accumulation between A. thaliana accessions. Only 13.4% of the mass peaks were detected in all 14 accessions analyzed. Quantitative trait locus (QTL) analysis of more than 2,000 mass peaks, detected in a recombinant inbred line (RIL) population derived from the two most divergent accessions, enabled the identification of QTLs for about 75% of the mass signals. More than one-third of the signals were not detected in either parent, indicating the large potential for modification of metabolic composition through classical breeding.

Antioxidants, phenolic compounds, and nutritional quality of different strawberry genotypes.

Strawberry contains high levels of micronutrients and phytochemical compounds. These exhibit functional roles in plant growth and metabolism and are also essential for the nutritional and organoleptic qualities of the fruit. The aim of the present work was to better characterize the phytochemical and antioxidant profiles of the fruit of nine different genotypes of strawberry, by measuring the total flavonoid, anthocyanin, vitamin C, and folate contents. Cultivar effects on the total antioxidant capacities of strawberries were also tested. In addition, the individual contribution of the main antioxidant compounds was assessed by HPLC separation coupled to an online postcolumn antioxidant detection system. This study showed the important role played by the genetic background on the chemical and antioxidant profiles of strawberry fruits. Significant differences were found between genotypes for the total antioxidant capacity and for all tested classes of compounds. The HPLC analyses confirmed qualitative and quantitative variability in the antioxidant profiles. These studies show that differences exist among cultivars, applicable in dietary studies in human subjects.

Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria x ananassa).

An octaploid (Fragaria x ananassa cv. Calypso) genotype of strawberry was transformed with an antisense chalcone synthase (CHS) gene construct using a ripening related CHS cDNA from Fragaria x ananassa cv. Elsanta under the control of the constitutive CaMV 35S promoter via Agrobacterium tumefaciens. Out of 25 transgenic lines, nine lines showed a reduction in CHS mRNA accumulation of more than 50% as compared to the untransformed cv. Calypso control. The antisense CHS construct was found to be integrated into the genome, with a copy number ranging from one to four. The pigmentation of the fruit was only affected when less than 5% of the control CHS expression level was detected. A stable antisense phenotype over a period of 4 years was obtained in the primary transgenic lines at a rate of 1:20. As a consequence of the reduced activity of CHS, the levels of anthocyanins, flavonols, and proanthocyanidins were downregulated and precursors of the flavonoid pathway were shunted to the phenylpropanoid pathway leading to highly increased levels of cinnamoyl glucose (520% of control), caffeoyl glucose (816% of control), and feruloyl glucose (1092% of control) as well as p-coumaryl alcohol (363% of control) and p-coumaryl-1-acetate (1079% of control), which occur only as trace components in untransformed control fruits. These results demonstrate that the introduction of an antisense CHS construct in strawberry results in an unpredictable biochemical phenotype, thereby confirming that CHS function is an important regulatory point of substrate flow between the flavonoid and the phenylpropanoid pathways.

Identification and dietary relevance of antioxidants from raspberry.

In this paper we review the current literature on antioxidants from fruit of red raspberry (Rubus idaeus) and place these in context concerning what is known from other food species. The review concentrates on the methods of antioxidant testing, the diversity of antioxidants in raspberry, effects of ripeness, cultivar, storage and processing techniques, and the bioavailability of raspberry antioxidants in humans after eating the fruit. It is clear that raspberry, like several other fruits and vegetables such as tomato, strawberry, kiwi and broccoli, represents a valuable contrasting source of potentially healthy compounds and can represent an important component of a balanced diet.

Unravelling enzymatic discoloration in potato through a combined approach of candidate genes, QTL, and expression analysis.

Enzymatic discoloration (ED) of potato tubers was investigated in an attempt to unravel the underlying genetic factors. Both enzyme and substrate concentration have been reported to influence the degree of discoloration and as such this trait can be regarded as polygenic. The diploid mapping population C x E, consisting of 249 individuals, was assayed for the degree of ED and levels of chlorogenic acid and tyrosine. Using this data, Quantitative Trait Locus (QTL) analysis was performed. Three QTLs for ED have been found on parental chromosomes C3, C8, E1, and E8. For chlorogenic acid a QTL has been identified on C2 and for tyrosine levels, a QTL has been detected on C8. None of the QTLs overlap, indicating the absence of genetic correlations between these components underlying ED, in contrast to earlier reports in literature. An obvious candidate gene for the QTL for ED on Chromosome 8 is polyphenol oxidase (PPO), which was previously mapped on chromosome 8. With gene-specific primers for PPO gene POT32 a CAPS marker was developed. Three different alleles (POT32-1, -2, and -3) could be discriminated. The segregating POT32 alleles were used to map the POT32 CAPS marker and QTL analysis was redone, showing that POT32 coincides with the QTL peak. A clear correlation between allele combinations and degree of discoloration was observed. In addition, analysis of POT32 gene expression in a subset of genotypes indicated a correlation between the level of gene expression and allele composition. On average, genotypes having two copies of allele 1 had both the highest degree of discoloration as well as the highest level of POT32 gene expression.

Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry (Fragaria xananassa).

The biosynthesis of flavonoids and proanthocyanidins was studied in cultivated strawberry (Fragaria xananassa) by combining biochemical and molecular approaches. Chemical analyses showed that ripe strawberries accumulate high amounts of pelargonidin-derived anthocyanins, and a larger pool of 3',4'-hydroxylated proanthocyanidins. Activities and properties of major recombinant enzymes were demonstrated by means of in vitro assays, with special emphasis on specificity for the biologically relevant 4'- and 3',4'-hydroxylated compounds. Only leucoanthocyanidin reductase showed a strict specificity for the 3',4'-hydroxylated leucocyanidin, while other enzymes accepted either hydroxylated substrate with different relative activity rates. The structure of late flavonoid pathway genes, leading to the synthesis of major compounds in ripe fruits, was elucidated. Complex developmental and spatial expression patterns were shown for phenylpropanoid and flavonoid genes in fruits throughout ripening as well as in leaves, petals and roots. Presented results elucidate key steps in the biosynthesis of strawberry flavonoid end products.

RNA interference silencing of chalcone synthase, the first step in the flavonoid biosynthesis pathway, leads to parthenocarpic tomato fruits.

Parthenocarpy, the formation of seedless fruits in the absence of functional fertilization, is a desirable trait for several important crop plants, including tomato (Solanum lycopersicum). Seedless fruits can be of great value for consumers, the processing industry, and breeding companies. In this article, we propose a novel strategy to obtain parthenocarpic tomatoes by down-regulation of the flavonoid biosynthesis pathway using RNA interference (RNAi)-mediated suppression of chalcone synthase (CHS), the first gene in the flavonoid pathway. In CHS RNAi plants, total flavonoid levels, transcript levels of both Chs1 and Chs2, as well as CHS enzyme activity were reduced by up to a few percent of the corresponding wild-type values. Surprisingly, all strong Chs-silenced tomato lines developed parthenocarpic fruits. Although a relation between flavonoids and parthenocarpic fruit development has never been described, it is well known that flavonoids are essential for pollen development and pollen tube growth and, hence, play an essential role in plant reproduction. The observed parthenocarpic fruit development appeared to be pollination dependent, and Chs RNAi fruits displayed impaired pollen tube growth. Our results lead to novel insight in the mechanisms underlying parthenocarpic fruit development. The potential of this technology for applications in plant breeding and biotechnology will be discussed.

Production of resveratrol in recombinant microorganisms.

Resveratrol production in Saccharomyces cerevisiae was compared to that in Escherichia coli. In both systems, 4-coumarate:coenzyme A ligase from tobacco and stilbene synthase from grapes were expressed. When p-coumaric acid was used as the precursor, resveratrol accumulations in the culture medium were observed to be comparable in E. coli (16 mg/liter) and yeast (6 mg/liter).

A liquid chromatography-mass spectrometry-based metabolome database for tomato.

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 alpha-tomatine were specifically present in the peel, while several other alkaloids and some particular phenylpropanoids were mainly present in the flesh tissue.

[Isolation and identification of glycinol from Glycine max [L.] Merri].

As one of the main phytoalexins and phytoestrogens, glyceollin is an important prenylflavonoid in Glycine max [L.] Merri. (soybean). Many kinds of elicitors can be used to induce its accumulation. Its biosynthesis pathway is commonly used to study the characteristics of prenyltransferase, which catalyzes the prenylated reaction happening in a very few plant families in nature. Glycinol, the direct precursor of glyceollin, is necessary to study the prenylated reaction in soybean. In comparing with the other elicitors to elicit the glycinol accumulation in soybean cotyledons, AgNO3 is the most effective elicitor. Exposure of 6-8 days old cotyledons to 0.01 mol/L AgNO3 and incubation for 24 h result in the accumulation of 256 microg (glycinol)/g (fresh weight). The glycinol was extracted by methanol. Then the isolation and purification were conducted by preparative high performance liquid chromatography. Instead of 100% acetonitrile-0.1% formic acid as the elution system, the extract was eluted by 100% methanol-0.1% formic acid. Glycinol eluted earlier than daidzin under this system and decreased the disturbance from the large amount of daidzin. Identification was performed by comparing the mass spectrum (liquid chromatography/quadrupole-time of flight) and ultraviolet spectrum with those of the standard. At last, 100 mg purified glycinol was obtained from 390 g of fresh material.

A novel approach for nontargeted data analysis for metabolomics. Large-scale profiling of tomato fruit volatiles.

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.