Annick Moing

Mapping QTLs controlling fruit quality in peach (Prunus persica (L.) Batsch)

Abstract The organoleptic quality of fleshy fruits is in a large part defined by their composition of soluble sugars and organic acids. An F2 population issuing from a cross between two peach varieties, ‘Ferjalou Jalousia’, a non-acid peach, and ‘Fantasia’, an acid nectarine, was analysed over 2 successive years for agronomic characters and for molecular-marker (isoenzymes, RFLPs, RAPDs, IMAs and AFLPs) segregations. Blooming and maturity dates, as well as productivity, were noted for each tree. Four fruits per tree were analysed at maturity for fresh weight, colour, pH, titratable acidity, soluble-solids content (SSC), acid (malic, citric and quinic acids) and sugar (sucrose, glucose, fructose, sorbitol) contents. QTLs were detected for all fruit components analysed, except for fruit colour. The QTLs for nearly all components were present on two linkage groups. For productivity, fresh weight, pH, quinic acid, sucrose and sorbitol content, all the detected QTLs displayed the same effect as the parental phenotypes. By contrast, for maturity date, titratable acidity, malic and citric acids and fructose, some QTLs displayed the same effect as the parental phenotypes while others displayed the opposite effect. The fraction of the total variation in each trait throughout the population explained by the QTLs was very high and reached more than 90% for some characters. For most of the characters analysed, epistasis was observed between QTLs.

Gene and Metabolite Regulatory Network Analysis of Early Developing Fruit Tissues Highlights New Candidate Genes for the Control of Tomato Fruit Composition and Development

Variations in early fruit development and composition may have major impacts on the taste and the overall quality of ripe tomato (Solanum lycopersicum) fruit. To get insights into the networks involved in these coordinated processes and to identify key regulatory genes, we explored the transcriptional and metabolic changes in expanding tomato fruit tissues using multivariate analysis and gene-metabolite correlation networks. To this end, we demonstrated and took advantage of the existence of clear structural and compositional differences between expanding mesocarp and locular tissue during fruit development (12–35 d postanthesis). Transcriptome and metabolome analyses were carried out with tomato microarrays and analytical methods including proton nuclear magnetic resonance and liquid chromatography-mass spectrometry, respectively. Pairwise comparisons of metabolite contents and gene expression profiles detected up to 37 direct gene-metabolite correlations involving regulatory genes (e.g. the correlations between glutamine, bZIP, and MYB transcription factors). Correlation network analyses revealed the existence of major hub genes correlated with 10 or more regulatory transcripts and embedded in a large regulatory network. This approach proved to be a valuable strategy for identifying specific subsets of genes implicated in key processes of fruit development and metabolism, which are therefore potential targets for genetic improvement of tomato fruit quality.

Candidate genes and QTLs for sugar and organic acid content in peach [Prunus persica (L.) Batsch]

Abstract.The identification of genes involved in variation of peach fruit quality would assist breeders in creating new cultivars with improved fruit quality. Major genes and quantitative trait loci (QTLs) for physical and chemical components of fruit quality have already been detected, based on the peach [Prunus persica (L.) Batsch] cv. Ferjalou Jalousia® (low-acid peach) × cv. Fantasia (normally-acid nectarine) F2 intraspecific cross. Our aim was to associate these QTLs to structural genes using a candidate gene/QTL approach. Eighteen cDNAs encoding key proteins in soluble sugar and organic acid metabolic pathways as well as in cell expansion were isolated from peach fruit. A single-strand conformation polymorphism strategy based on specific cDNA-based primers was used to map the corresponding genes. Since no polymorphism could be detected in the Ferjalou Jalousia® × Fantasia population, gene mapping was performed on the almond [Prunus amygdalus (P. dulcis)] cv. Texas × peach cv. Earlygold F2 interspecific cross from which a saturated map was available. Twelve candidate genes were assigned to four linkage groups of the peach genome. In a second step, the previous QTL detection was enhanced by integrating anchor loci between the Ferjalou Jalousia® × Fantasia and Texas × Earlygold maps and data from a third year of trait assessment on the Ferjalou Jalousia® × Fantasia population. Comparative mapping allowed us to detect a candidate gene/QTL co-location. It involved a cDNA encoding a vacuolar H+-pyrophosphatase (PRUpe;Vp2) that energises solute accumulation, and QTLs for sucrose and soluble solid content. This preliminary result may be the first step in the future development of marker-assisted selection for peach fruit sucrose and soluble solid content.

Quantitative metabolic profiling by 1-dimensional 1H-NMR analyses: application to plant genetics and functional genomics

Metabolic profiling by 1-dimensional (1-D) 1H-nuclear magnetic resonance (NMR) was tested for absolute quantification of soluble sugars, organic acids, amino acids and some secondary metabolites in fruit, roots and leaves. The metabolite responsible for each peak of the 1H-NMR spectra was identified from spectra of pure compounds. Peak identity was confirmed by the addition of a small amount of commercially-available pure substance. 1H-NMR spectra acquisition was automated. 1H-NMR absolute quantification was performed with a synthesised electronic reference signal and validated by comparison with enzymatic or HPLC analyses; the correlation coefficients between 1H-NMR data and enzymatic or HPLC data were highly significant. Depending on the species and tissues, 14-17 metabolites could be quantified with 15-25 min acquisition time. The detection limit was approximately 1-9 µg in the NMR tube, depending on the compound. Quantitative data were used for (1) a genetic study of strawberry fruit quality, (2) a functional study of tomato transformants overexpressing hexokinase and (3) a study of Arabidopsis phosphoenolpyruvate carboxylase transformants with several lines showing decreased activity of the enzyme. Biochemical phenotyping of the fruits of a strawberry offspring allowed the detection of quantitative trait loci (QTL) controlling fruit quality. Comparison of the roots of wild types and hexokinase tomato transformants using principal component analysis of metabolic profiles revealed that environmental factors, i.e. culture conditions, can significantly modify the metabolic status of plants and thus hide or emphasise the expression of a given genetic background. The decrease in phosphoenolpyruvate carboxylase activity (up to 75%) in Arabidopsis transformants impacted on the metabolic profiles without compromising plant growth, thus supporting the idea that the enzyme has a low influence on the carbon flux through the anaplerotic pathway.

1H NMR, GC-EI-TOFMS, and data set correlation for fruit metabolomics: Application to spatial metabolite analysis in melon

A metabolomics approach combining (1)H NMR and gas chromatography-electrospray ionization time-of-flight mass spectrometry (GC-EI-TOFMS) profiling was employed to characterize melon (Cucumis melo L.) fruit. In a first step, quantitative (1)H NMR of polar extracts and principal component analyses (PCA) of the corresponding data highlighted the major metabolites in fruit flesh, including sugars, organic acids, and amino acids. In a second step, the spatial localization of metabolites was investigated using both analytical techniques. Direct (1)H NMR profiling of juice or GC-EI-TOFMS profiling of tissue extracts collected from different locations in the fruit flesh provided information on advantages and drawbacks of each technique for the analysis of a sugar-rich matrix such as fruit. (1)H NMR and GC-EI-TOFMS data sets were compared using independently performed PCA and multiblock hierarchical PCA (HPCA), respectively. In addition a correlation-based multiblock HPCA was used for direct comparison of both analytical data sets. These data analyses revealed several gradients of metabolites in fruit flesh which can be related with differences in metabolism and indicated the suitability of multiblock HPCA for correlation of data from two (or potentially more) metabolomics platforms.

Extensive metabolic cross-talk in melon fruit revealed by spatial and developmental combinatorial metabolomics

• Variations in tissue development and spatial composition have a major impact on the nutritional and organoleptic qualities of ripe fleshy fruit, including melon (Cucumis melo). To gain a deeper insight into the mechanisms involved in these changes, we identified key metabolites for rational food quality design. • The metabolome, volatiles and mineral elements were profiled employing an unprecedented range of complementary analytical technologies. Fruits were followed at a number of time points during the final ripening process and tissues were collected across the fruit flesh from rind to seed cavity. Approximately 2000 metabolite signatures and 15 mineral elements were determined in an assessment of temporal and spatial melon fruit development. • This study design enabled the identification of: coregulated hubs (including aspartic acid, 2-isopropylmalic acid, β-carotene, phytoene and dihydropseudoionone) in metabolic association networks; global patterns of coordinated compositional changes; and links of primary and secondary metabolism to key mineral and volatile fruit complements. • The results reveal the extent of metabolic interactions relevant to ripe fruit quality and thus have enabled the identification of essential candidate metabolites for the high-throughput screening of melon breeding populations for targeted breeding programmes aimed at nutrition and flavour improvement.

Non-structural carbohydrates in woody plants compared among laboratories

Non-structural carbohydrates (NSC) in plant tissue are frequently quantified to make inferences about plant responses to environmental conditions. Laboratories publishing estimates of NSC of woody plants use many different methods to evaluate NSC. We asked whether NSC estimates in the recent literature could be quantitatively compared among studies. We also asked whether any differences among laboratories were related to the extraction and quantification methods used to determine starch and sugar concentrations. These questions were addressed by sending sub-samples collected from five woody plant tissues, which varied in NSC content and chemical composition, to 29 laboratories. Each laboratory analyzed the samples with their laboratory-specific protocols, based on recent publications, to determine concentrations of soluble sugars, starch and their sum, total NSC. Laboratory estimates differed substantially for all samples. For example, estimates for Eucalyptus globulus leaves (EGL) varied from 23 to 116 (mean = 56) mg g(-1) for soluble sugars, 6-533 (mean = 94) mg g(-1) for starch and 53-649 (mean = 153) mg g(-1) for total NSC. Mixed model analysis of variance showed that much of the variability among laboratories was unrelated to the categories we used for extraction and quantification methods (method category R(2) = 0.05-0.12 for soluble sugars, 0.10-0.33 for starch and 0.01-0.09 for total NSC). For EGL, the difference between the highest and lowest least squares means for categories in the mixed model analysis was 33 mg g(-1) for total NSC, compared with the range of laboratory estimates of 596 mg g(-1). Laboratories were reasonably consistent in their ranks of estimates among tissues for starch (r = 0.41-0.91), but less so for total NSC (r = 0.45-0.84) and soluble sugars (r = 0.11-0.83). Our results show that NSC estimates for woody plant tissues cannot be compared among laboratories. The relative changes in NSC between treatments measured within a laboratory may be comparable within and between laboratories, especially for starch. To obtain comparable NSC estimates, we suggest that users can either adopt the reference method given in this publication, or report estimates for a portion of samples using the reference method, and report estimates for a standard reference material. Researchers interested in NSC estimates should work to identify and adopt standard methods.

Effects of long-term cadmium exposure on growth and metabolomic profile of tomato plants

The response of tomato plants to long-term cadmium exposure was evaluated after a 90-days long culture in hydroponic conditions (0, 20, and 100 μM CdCl(2)). Cadmium preferentially accumulated in roots, and to a lower extent in upper parts of plants. Absolute quantification of 28 metabolites was obtained through (1)H NMR, HPLC-PDA, and colorimetric methods. The principal component analysis showed a clear separation between control and Cd treated samples. Proline and total ascorbate amounts were reduced in Cd-treated leaves, whereas α-tocopherol, asparagine, and tyrosine accumulation increased, principally in 100 μM Cd treated leaves. Carotenoid and chlorophyll contents decreased only in 100 μM Cd-mature-leaves, which correlate with a reduced expression of genes essential for isoprenoid and carotenoid accumulations. Our results show that tomato plants acclimatize during long-term exposure to 20 μM Cd. On the contrary, 100μM Cd treatment results in drastic physiological and metabolic perturbations leading to plant growth limitation and fruit set abortion.

Sugar Import and Phytopathogenicity of Spiroplasma citri: Glucose and Fructose Play Distinct Roles

We have shown previously that the glucose PTS (phosphotransferase system) permease enzyme II of Spiroplasma citri is split into two distinct polypeptides, which are encoded by two separate genes, crr and ptsG. A S. citri mutant was obtained by disruption of ptsG through homologous recombination and was proved unable to import glucose. The ptsG mutant (GII3-glc1) was transmitted to periwinkle (Catharanthus roseus) plants through injection to the leaf-hopper vector. In contrast to the previously characterized fructose operon mutant GMT 553, which was found virtually nonpathogenic, the ptsG mutant GII3-glc1 induced severe symptoms similar to those induced by the wild-type strain GII-3. These results, indicating that fructose and glucose utilization were not equally involved in pathogenicity, were consistent with biochemical data showing that, in the presence of both sugars, S. citri used fructose preferentially. Proton nuclear magnetic resonance analyses of carbohydrates in plant extracts revealed the accumulation of soluble sugars, particularly glucose, in plants infected by S. citri GII-3 or GII3-glc1 but not in those infected by GMT 553. From these data, a hypothetical model was proposed to establish the relationship between fructose utilization by the spiroplasmas present in the phloem sieve tubes and glucose accumulation in the leaves of S. citri infected plants.

Genetic dissection of fruit quality traits in the octoploid cultivated strawberry highlights the role of homoeo-QTL in their control

Fruit quality traits are major breeding targets in the Rosaceae. Several of the major Rosaceae species are current or ancient polyploids. To dissect the inheritance of fruit quality traits in polyploid fleshy fruit species, we used a cultivated strawberry segregating population comprising a 213 full-sibling F1 progeny from a cross between the variety ‘Capitola’ and the genotype ‘CF1116’. We previously developed the most comprehensive strawberry linkage map, which displays seven homoeology groups (HG), including each four homoeology linkage groups (Genetics 179:2045–2060, 2008). The map was used to identify quantitative trait loci (QTL) for 19 fruit traits related to fruit development, texture, colour, anthocyanin, sugar and organic acid contents. Analyses were carried out over two or three successive years on field-grown plants. QTL were detected for all the analysed traits. Because strawberry is an octopolyploid species, QTL controlling a given trait and located at orthologous positions on different homoeologous linkage groups within one HG are considered as homoeo-QTL. We found that, for various traits, about one-fourth of QTL were putative homoeo-QTL and were localised on two linkage groups. Several homoeo-QTL could be detected the same year, suggesting that several copies of the gene underlying the QTL are functional. The detection of some other homoeo-QTL was year-dependent. Therefore, changes in allelic expression could take place in response to environmental changes. We believe that, in strawberry as in other polyploid fruit species, the mechanisms unravelled in the present study may play a crucial role in the variations of fruit quality.