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PtdIns is an important precursor for inositol-containing lipids, including polyphosphoinositides, which have multiple essential functions in eukaryotic cells. It was previously proposed that different regulatory functions of inositol-containing lipids may be performed by independent lipid pools; however, it remains unclear how such subcellular pools are established and maintained. In the present paper, a previously uncharacterized Arabidopsis gene product with similarity to the known Arabidopsis PIS (PtdIns synthase), PIS1, is shown to be an active enzyme, PIS2, capable of producing PtdIns in vitro. PIS1 and PIS2 diverged slightly in substrate preferences for CDP-DAG [cytidinediphospho-DAG (diacylglycerol)] species differing in fatty acid composition, PIS2 preferring unsaturated substrates in vitro. Transient expression of fluorescently tagged PIS1 or PIS2 in onion epidermal cells indicates localization of both enzymes in the ER (endoplasmic reticulum) and, possibly, Golgi, as was reported previously for fungal and mammalian homologues. Constitutive ectopic overexpression of PIS1 or PIS2 in Arabidopsis plants resulted in elevated levels of PtdIns in leaves. PIS2-overexpressors additionally exhibited significantly elevated levels of PtdIns(4)P and PtdIns(4,5)P(2), whereas polyphosphoinositides were not elevated in plants overexpressing PIS1. In contrast, PIS1-overexpressors contained significantly elevated levels of DAG and PtdEtn (phosphatidylethanolamine), an effect not observed in plants overexpressing PIS2. Biochemical analysis of transgenic plants with regards to fatty acids associated with relevant lipids indicates that lipids increasing with PIS1 overexpression were enriched in saturated or monounsaturated fatty acids, whereas lipids increasing with PIS2 overexpression, including polyphosphoinositides, contained more unsaturated fatty acids. The results indicate that PtdIns populations originating from different PIS isoforms may enter alternative routes of metabolic conversion, possibly based on specificity and immediate metabolic context of the biosynthetic enzymes.

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Solanesol is a high value 45-carbon, unsaturated, all-trans-nonaprenol isoprenoid. Recently solanesol has received particular attention because of its utility, both in its own right and as a precursor in the production of numerous compounds used in the treatment of disease states. Solanesol is found mainly in solanaceous crops such as potato, tomato, tobacco and pepper where it accumulates in the foliage. There is considerable potential to explore the extraction of solanesol from these sources as a valuable co-product. In this study we have characterized the genetic variation in leaf solanesol content in a biparental, segregating diploid potato population. We demonstrate that potato leaf solanesol content is genetically controlled and identify several quantitative trait loci associated with leaf solanesol content. Transient over-expression of genes from the methylerythritol 4-phosphate (MEP) and mevalonic acid (MVA) pathways, either singly or in combination, resulted in enhanced accumulation of solanesol in leaves of Nicotiana benthamiana, providing insights for genetically engineering the pathway. We also demonstrate that in potato, leaf solanesol content is enhanced by up to six-fold on exposure to moderately elevated temperature and show corresponding changes in expression patterns of MEP and MVA genes. Our combined approaches offer new insights into solanesol accumulation and strategies for developing a bio-refinery approach to potato production.

IntroductionBlackcurrant (Ribes nigrum L.) is an excellent example of a “super fruit” with potential health benefits. Both genotype and cultivation environment are known to affect the chemical composition of blackcurrant, especially ascorbic acid and various phenolic compounds. Environmental conditions, like temperature, solar radiation and precipitation can also have significant impact on fruit chemical composition. The relevance of the study is further accentuated by the predicted and ongoing changes in global climate.ObjectivesThe aim of the present study was to provide new knowledge and a deeper understanding of the effects of post flowering environmental conditions, namely temperature and day length, on fruit quality and chemical composition of blackcurrant using an untargeted high performance liquid chromatography–photo diode array–mass spectrometry (HPLC–PDA–MS) metabolomics approach.MethodsA phytotron experiment with cultivation of single-stemmed potted plants of blackcurrant cv. Narve Viking was conducted using constant temperatures of 12, 18 or 24 °C and three different photoperiods (short day, short day with night interruption, and natural summer daylight conditions). Plants were also grown under ambient outdoor conditions. Ripe berries were analysed using an untargeted HPLC–PDA–MS metabolomics approach to detect the presence and concentration of molecules as affected by controlled climatic factors.ResultsThe untargeted metabolomics dataset contained a total of 7274 deconvolved retention time-m/z pairs across both electrospray ionisation (ESI) positive and negative polarities, from which 549 metabolites were identified or minimally annotated based upon accurate mass MS. Conventional principal component analysis (PCA) in combination with the Friedman significance test were applied to first identify which metabolites responded to temperature in a linear fashion. Multi-block hierarchical PCA in combination with the Friedman significance test was secondly applied to identify metabolites that were responsive to different day length conditions. Temperature had significant effect on a total of 365 metabolites representing a diverse range of chemical classes. It was observed that ripening of the blackcurrant berries under ambient conditions, compared to controlled conditions, resulted in an increased accumulation of 34 annotated metabolites, mainly anthocyanins and flavonoids. 18 metabolites were found to be regulated differentially under the different daylength conditions. Moreover, based upon the most abundant anthocyanins, a comparison between targeted and untargeted analyses, revealed a close convergence of the two analytical methods. Therefore, the study not just illustrates the value of non-targeted metabolomics approaches with respect to the huge diversity and numbers of significantly changed metabolites detected (and which would be missed by conventional targeted analyses), but also shows the validity of the non-targeted approach with respect to its precision compared to targeted analyses.ConclusionsBla...

BackgroundFlavonoids are produced in all flowering plants in a wide range of tissues including in berry fruits. These compounds are of considerable interest for their biological activities, health benefits and potential pharmacological applications. However, transcriptomic and genomic resources for wild and cultivated berry fruit species are often limited, despite their value in underpinning the in-depth study of metabolic pathways, fruit ripening as well as in the identification of genotypes rich in bioactive compounds.ResultsTo access the genetic diversity of wild and cultivated berry fruit species that accumulate high levels of phenolic compounds in their fleshy berry(-like) fruits, we selected 13 species from Europe, South America and Asia representing eight genera, seven families and seven orders within three clades of the kingdom Plantae. RNA from either ripe fruits (ten species) or three ripening stages (two species) as well as leaf RNA (one species) were used to construct, assemble and analyse de novo transcriptomes. The transcriptome sequences are deposited in the BacHBerryGEN database (http://jicbio.nbi.ac.uk/berries) and were used, as a proof of concept, via its BLAST portal (http://jicbio.nbi.ac.uk/berries/blast.html) to identify candidate genes involved in the biosynthesis of phenylpropanoid compounds. Genes encoding regulatory proteins of the anthocyanin biosynthetic pathway (MYB and basic helix-loop-helix (bHLH) transcription factors and WD40 repeat proteins) were isolated using the transcriptomic resources of wild blackberry (Rubus genevieri) and cultivated red raspberry (Rubus idaeus cv. Prestige) and were shown to activate anthocyanin synthesis in Nicotiana benthamiana. Expression patterns of candidate flavonoid gene transcripts were also studied across three fruit developmental stages via the BacHBerryEXP gene expression browser (http://www.bachberryexp.com) in R. genevieri and R. idaeus cv. Prestige.ConclusionsWe report a transcriptome resource that includes data for a wide range of berry(-like) fruit species that has been developed for gene identification and functional analysis to assist in berry fruit improvement. These resources will enable investigations of metabolic processes in berries beyond the phenylpropanoid biosynthetic pathway analysed in this study. The RNA-seq data will be useful for studies of berry fruit development and to select wild plant species useful for plant breeding purposes.

The effect of photoperiod on nutritional quality and chemical composition of 'Glen Ample' red raspberry has been studied under controlled environment conditions. Long day (LD) conditions during fruit growth and maturation significantly increased fruit concentrations of ascorbic acid, total phenolics, organic acids such as malic, quinic and ellagic acids, as well as the ferric reducing activity power, while reducing the concentrations of soluble solids, sucrose and the sugar/acid ratio. The levels of total anthocyanin and oxalic acid were not significantly affected by photoperiod whereas semi-quantitative analysis indicated a significant increase in the levels of some ellagitannins. The same responses were found whether LD was established by day-length extension with low-intensity light or by night interruption, thus demonstrating that the observed changes in nutritional quality of raspberry fruit was mediated through specific photoperiodic mechanism(s) independently of the daily light energy supply.

Plants are a reservoir of high-value molecules with underexplored biomedical applications. With the aim of identifying novel health-promoting attributes in underexplored natural sources, we scrutinized the diversity of (poly)phenols present within the berries of selected germplasm from cultivated, wild, and underutilized Rubus species. Our strategy combined the application of metabolomics, statistical analysis, and evaluation of (poly)phenols’ bioactivity using a yeast-based discovery platform. We identified species as sources of (poly)phenols interfering with pathological processes associated with redox-related diseases, particularly, amyotrophic lateral sclerosis, cancer, and inflammation. In silico prediction of putative bioactives suggested cyanidin–hexoside as an anti-inflammatory molecule which was validated in yeast and mammalian cells. Moreover, cellular assays revealed that the cyanidin moiety was responsible for the anti-inflammatory properties of cyanidin–hexoside. Our findings unveiled novel (poly)phenolic bioactivities and illustrated the power of our integrative approach for the identification of dietary (poly)phenols with potential biomedical applications.