MSClust: a tool for unsupervised mass spectra extraction of chromatography-mass spectrometry ion-wise aligned data

Mass peak alignment (ion-wise alignment) has recently become a popular method for unsupervised data analysis in untargeted metabolic profiling. Here we present MSClust—a software tool for analysis GC–MS and LC–MS datasets derived from untargeted profiling. MSClust performs data reduction using unsupervised clustering and extraction of putative metabolite mass spectra from ion-wise chromatographic alignment data. The algorithm is based on the subtractive fuzzy clustering method that allows unsupervised determination of a number of metabolites in a data set and can deal with uncertain memberships of mass peaks in overlapping mass spectra. This approach is based purely on the actual information present in the data and does not require any prior metabolite knowledge. MSClust can be applied for both GC–MS and LC–MS alignment data set

The mQTL hotspot on linkage group 16 for phenolic compounds in apple fruits is probably the result of a leucoanthocyanidin reductase gene at that locus

BACKGROUND: Our previous study on ripe apples from a progeny of a cross between the apple cultivars 'Prima' and 'Fiesta' showed a hotspot of mQTLs for phenolic compounds at the top of LG16, both in peel and in flesh tissues. In order to find the underlying gene(s) of this mQTL hotspot, we investigated the expression profiles of structural and putative transcription factor genes of the phenylpropanoid and flavonoid pathways during different stages of fruit development in progeny genotypes. RESULTS: Only the structural gene leucoanthocyanidin reductase (MdLAR1) showed a significant correlation between transcript abundance and content of metabolites that mapped on the mQTL hotspot. This gene is located on LG16 in the mQTL hotspot. Progeny that had inherited one or two copies of the dominant MdLAR1 alleles (Mm, MM) showed a 4.4- and 11.8-fold higher expression level of MdLAR1 respectively, compared to the progeny that had inherited the recessive alleles (mm). This higher expression was associated with a four-fold increase of procyanidin dimer II as one representative metabolite that mapped in the mQTL hotspot. Although expression level of several structural genes were correlated with expression of other structural genes and with some MYB and bHLH transcription factor genes, only expression of MdLAR1 was correlated with metabolites that mapped at the mQTL hotspot.MdLAR1 is the only candidate gene that can explain the mQTL for procyanidins and flavan-3-ols. However, mQTLs for other phenylpropanoids such as phenolic esters, dihydrochalcones and flavonols, that appear to map at the same locus, have so far not been considered to be dependent on LAR, as their biosynthesis does not involve LAR activity. An explanation for this phenomenon is discussed. CONCLUSIONS: Transcript abundances and genomic positions indicate that the mQTL hotspot for phenolic compounds at the top of LG16 is controlled by the MdLAR1 gene. The dominant allele of the MdLAR1 gene, causing increased content of metabolites that are potentially health beneficial, could be used in marker assisted selection of current apple breeding programs and for cisgenesi

Clinical Studies into the Causes of Idiopathic Macular Telangiectasia Type 2: Sleep Apnoea and Macular Telangiectasia: The SAMTel Project

Purpose: To assess the prevalence of Obstructive Sleep Apnoea (OSA) in a population with Macular Telangiectasia Type 2 (MacTel) and how OSA impacts on MacTel progression. Methods: In this case-control study participants completed a questionnaire which incorporated the Berlin Questionnaire (BQ) and questions regarding anthropometric data and medical history. A subset was sequentially selected to undertake overnight sleep analysis using the ResMed ApneaLink™. Using data acquired from the Busselton Population and Medical Research Foundation participants were case-matched based on age, sex and body mass index (BMI) along with, where possible, the presence of hypertension and diabetes. Results: There were 57 (30 ApneaLink) MacTel and 183 controls, respectively. There was no difference in self-reported sleep disordered breathing outcomes between the cohorts using the BQ (p=0.95). Analysis of key indices from ApneaLink recordings found that those with an Apnoea – Hypopnoea Index (AHI) and Oxygen Desaturation Index (ODI) > 5 episodes per hour had a more advanced stage of MacTel (AHI p = 0.05, ODI p = 0.03). 2 year MacTel disease progression rates were unremarkable. Conclusion: Patients with MacTel have a high prevalence of OSA which appears to result in a more advanced form of the disease

Virus-induced gene silencing in detached tomatoes and biochemical effects of phytoene desaturase gene silencing

Virus-induced gene silencing (VIGS) is a technology that has rapidly emerged for gene function studies in plants. Many advances have been made in applying this technique in an increasing number of crops. Recently, VIGS has been successfully used to silence genes in tomato fruit through agroinfiltration of fruit attached to the plant. The phytoene desaturase (Pds) gene has been widely used as a reporter gene in VIGS experiments, although little is known about the changes that occur due to its silencing in plants. In this paper, we describe the efficient silencing of the Pds gene through the VIGS approach in detached tomato fruits, which makes the VIGS procedure even more versatile and applicable. After 16 days of agroinfiltration, approximately 75% of the tomatoes showed Pds silencing symptoms, although the distribution of silenced areas was variable among fruits. To study the potential effects caused by Pds silencing in detached tomatoes, carotenoids and other semi-polar secondary metabolites were analyzed using Liquid Chromatography-Mass Spectrometry. In addition, potential differences in primary metabolites were analyzed using Gas Chromatography-Mass Spectrometry. The results indicated that the yellow phenotype observed in Pds-silenced fruit was mainly due to the lack of the red-colored lycopene and therefore to a more pronounced contribution of the yellow-orange carotenoids (lutein, violaxanthin, and zeaxanthin) to the final color of the fruits. Furthermore, the biochemical changes observed in Pds-silenced detached tomatoes suggested that carotenoid and other pathways, e.g. leading to alkaloids and flavonoids, might be affected by the silencing of this reporter gene, and this should be taken into consideration for future experimental designs

Application of ISSR markers in the genus Lycopersicon

The level of polymorphism in tomato was studied using ISSR-PCR. Five tomato species: Lycopersicon esculentum, Lycopersicon pennellii, Lycopersicon cheesmanii, Lycopersicon humboldtii, Lycopersicon hirsutum and two Lycopersicon esculentum substitution lines IL 6-3 and WSL 6 were analyzed. ISSR-PCR was performed with fourteen primers. Nine of these fourteen primers were individually able to distinguish all tomato species. The data were used to create a phylogenetic tree of the five tomato species. The tree showed complete correspondence to previous phylogenetic investigations. ISSR-PCR on two Lycopersicon esculentum substitution lines IL 6-3 and WSL 6 enabled us to place thirteen ISSR markers on the classical map of Lycopersicon esculentum chromosome 6. Some of the markers were not located in the pericentromeric region. Using one ISSR and one RGA (Resistance-gene analogs) primer resulted in fingerprints having some new bands compared with ISSR fingerprints

Characterization of volatile and non-volatile compounds of fresh pepper (Capsicum annuum)

In this study volatile and non-volatile compounds and several agronomical important parameters were measured in mature fruits of elite sweet pepper breeding lines and hybrids and several genebank accessions from different Capsicum species. The sweet pepper breeding lines and hybrids were chosen to roughly represent the expected variation in flavor of Capsicum annuum in the Rijk Zwaan germplasm. The genebank accessions were either chosen because they were expected to have unique combinations of aromas and flavors, according to experience and/or literature, or were parents of mapping populations. The biochemical profiling allowed visualization of between- and within-species metabolic variation and stability during the year. In general, total soluble solids content (Brix) was genotype-dependent and fluctuated only slightly throughout the growing season, with uncultivated genotypes showing the largest changes. The species C. chinense, C. baccatum var. pendulum and C. annuum could be clearly separated by principle component analysis based on profiles of 391 volatile compounds. Especially for breeding purposes it seems to be interesting to study this variation in more detail, trying to unravel the complex genetics of the different pepper flavor aspects

Solanum lycopersicum AUXIN RESPONSE FACTOR 9 regulates cell division activity during early tomato fruit development

The transformation of the ovary into a fruit after successful completion of pollination and fertilization has been associated with many changes at transcriptomic level. These changes are part of a dynamic and complex regulatory network that is controlled by phytohormones, with a major role for auxin. One of the auxin-related genes differentially expressed upon fruit set and early fruit development in tomato is Solanum lycopersicum AUXIN RESPONSE FACTOR 9 (SlARF9). Here, the functional analysis of this ARF is described. SlARF9 expression was found to be auxin-responsive and SlARF9 mRNA levels were high in the ovules, placenta, and pericarp of pollinated ovaries, but also in other plant tissues with high cell division activity, such as the axillary meristems and root meristems. Transgenic plants with increased SlARF9 mRNA levels formed fruits that were smaller than wild-type fruits because of reduced cell division activity, whereas transgenic lines in which SlARF9 mRNA levels were reduced showed the opposite phenotype. The expression analysis, together with the phenotype of the transgenic lines, suggests that, in tomato, ARF9 negatively controls cell division during early fruit development.</p

A taste of sweet pepper: Volatile and non-volatile chemical composition of fresh sweet pepper (Capsicum annuum) in relation to sensory evaluation of taste

In this study volatile and non-volatile compounds, as well as some breeding parameters, were measured in mature fruits of elite sweet pepper (Capsicum annuum) lines and hybrids from a commercial breeding program, several cultivated genotypes and one gene bank accession. In addition, all genotypes were evaluated for taste by a trained descriptive sensory expert panel. Metabolic contrasts between genotypes were caused by clusters of volatile and non-volatile compounds, which could be related to metabolic pathways and common biochemical precursors. Clusters of phenolic derivatives, higher alkanes, sesquiterpenes and lipid derived volatiles formed the major determinants of the genotypic differences. Flavour was described with the use of 14 taste attributes, of which the texture related attributes and the sweet-sour contrast were the most discriminatory factors. The attributes juiciness, toughness, crunchiness, stickiness, sweetness, aroma, sourness and fruity/apple taste could be significantly predicted with combined volatile and non-volatile data. Fructose and (E)-2-hexen-1-ol were highly correlated with aroma, fruity/apple taste and sweetness. New relations were found for fruity/apple taste and sweetness with the compounds p-menth-1-en-9-al, (E)-beta-ocimene, (Z)-2-penten-1-ol and (E)-geranylacetone. Based on the overall biochemical and sensory results, the perspectives for flavour improvement by breeding are discussed

Low Salicylic Acid Level Improves Pollen Development Under Long-Term Mild Heat Conditions in Tomato

Exposure to high temperatures leads to failure in pollen development, which may have significant implications for food security with ongoing climate change. We hypothesized that the stress response-associated hormone salicylic acid (SA) affects pollen tolerance to long-term mild heat (LTMH) (≥14 days exposure to day-/nighttime temperature of 30–34/24–28°C, depending on the genotype), either positively, by inducing acclimation, or negatively, by reducing investment in reproductive development. Here, we investigated these hypotheses assessing the pollen thermotolerance of a 35S:nahG tomato line, which has low SA levels. We found that reducing the SA level resulted in increased pollen viability of plants grown in LTMH and further characterized this line by transcriptome, carbohydrate, and hormone analyses. Low expression of JAZ genes in 35S:nahG and LTMH hypersensitivity of low-jasmonic acid (JA) genotypes together suggest that the increased pollen thermotolerance in the low-SA line involves enhanced JA signal in developing anthers in LTMH. These findings have potential application in the development of more thermotolerant crops