Dataset Information

Application of HPLC-PDA-MS metabolite profiling to investigate the effect of growth temperature and day length on blackcurrant fruit

ABSTRACT: INTRODUCTION: Blackcurrant (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.
OBJECTIVES: The 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.
METHODS: A 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.
RESULTS: The 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.
CONCLUSIONS: Blackcurrant maturation under controlled ambient conditions revealed a number of insightful relationships between environment and chemical composition of the fruit. A prominent reduction of the most abundant anthocyanins under the highest temperature treatments indicated that blackcurrant berries in general may accumulate lower total anthocyanins in years with extreme hot summer conditions. HPLC–PDA–MS metabolomics is an excellent method for broad analysis of chemical composition of berries rich in phenolic compounds. Moreover, the experiment in controlled phytotron conditions provided additional knowledge concerning plant interactions with the environment.

INSTRUMENT(S): Liquid Chromatography MS - Negative (LC-MS (Negative)), Liquid Chromatography Tandem MS (LC-MS/MS), Liquid Chromatography MS - Positive (LC-MS (Positive))

SUBMITTER: James William Allwood

PROVIDER: MTBLS773 | MetaboLights | 2019-02-12

REPOSITORIES: MetaboLights

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			Action	DRS
	MTBLS773	Other
	FILES	Other
	a_MTBLS773_MS2_MS3_mass_spectrometry.txt	Txt
	a_MTBLS773_NEG_mass_spectrometry.txt	Txt
	a_MTBLS773_POS_mass_spectrometry.txt	Txt

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Project description:INTRODUCTION: Blackcurrant (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. </br> OBJECTIVES: The 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. </br> METHODS: A 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. </br> RESULTS: The 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. </br> CONCLUSIONS: Blackcurrant maturation under controlled ambient conditions revealed a number of insightful relationships between environment and chemical composition of the fruit. A prominent reduction of the most abundant anthocyanins under the highest temperature treatments indicated that blackcurrant berries in general may accumulate lower total anthocyanins in years with extreme hot summer conditions. HPLC–PDA–MS metabolomics is an excellent method for broad analysis of chemical composition of berries rich in phenolic compounds. Moreover, the experiment in controlled phytotron conditions provided additional knowledge concerning plant interactions with the environment.

Project description:Anthocyanins are flavonoid compounds responsible for red/purple colours in the leaves, fruit and flowers of many plant species. They are produced through a multistep pathway which is controlled by MYB transcription factors. VvMYBA1 and VvMYBA2 activate anthocyanin biosynthesis in grapevine (Vitis vinifera) and are non-functional in white grapevine cultivars. In this study, transgenic grapevines with altered VvMYBA gene expression were developed, and transcript analysis was carried out on berries using a microarray technique. The results showed that VvMYBA is a positive regulator of the later stages of anthocyanin synthesis, modification and transport in Shiraz. One up-regulated gene ANTHOCYANIN 3- O-GLUCOSIDE-6â-O-ACYLTRANSFERASE (Vv3AT) encodes a BAHD acyltransferase protein, belonging to a clade separate from most anthocyanin acyltransferases. Functional studies (in planta and in vitro) show that Vv3AT has a broad anthocyanin substrate specificity and can also utilise both aliphatic and aromatic acyl donors, a novel activity for this enzyme family found in nature. In V. vinifera cv. Pinot Noir, a red-berried grapevine mutant lacking acylated anthocyanins, Vv3AT contains a nonsense mutation encoding a truncated protein that lacks two motifs required for BAHD protein activity. Promoter activation assays confirm that Vv3AT transcription is activated by VvMYBA1, which adds to the current understanding of the regulation of the BAHD gene family. The flexibility of Vv3AT to use both classes of acyl donors will be useful in the engineering of anthocyanins in planta or in vitro. Transgenic Chardonnay/Shiraz and non-transformed WT controls were all grown in the same glasshouse in ambient light with a night break. Day and night temperatures were approximately 27oC and 22oC respectively. For microarray experiments, whole berries were sampled from independent transgenic lines: three from transgenic Chardonnay and four from transgenic Shiraz, resulting in three and four biological replicates respectively. Bunches were harvested close to ripeness based on average total soluble solids (TSS, measured as oBrix). This was aimed to be between 20 â 24 oBrix and was determined from TTS of a subsamples from each bunch (Table S53). A sample consisted of all remaining berries from a single bunch except when there were <100 berries in which case more than one bunch was used in the one replicate. Whole berries were immediately frozen in liquid nitrogen. For skin samples, the skins were first removed from fresh berries then frozen in liquid nitrogen. All samples were stored at -80oC.

Project description:Studying the effect of varying durations (1, 7 and 14 days) of locally applied heat stress on grape berries at three developmental stages (middle green, veraison and middle ripening). Among various environmental factors, temperature is a major regulator affecting plant growth, development and fruit composition. Reproductive development of grapevine and berry composition, are both highly influenced by temperature. To date, the molecular mechanisms involved in grapevine berries tolerance to high temperatures (HS) are poorly understood. Although recent data addressed the consequences on berry development of elevated temperatures applied at the whole plant level, the present work particularly focuses on the fruit responses triggered by direct exposure to heat stress. In the context of climate change, this work focused on microclimate effect is of particular interest to better understand the consequences of leaf removal (a common viticultural practice) on berry development. HS (+ 8°C) was locally applied to clusters from Cabernet Sauvignon fruiting cuttings at three different developmental stages (middle green, véraison and middle ripening). Samples were collected 1, 7 and 14 days after treatment and used for metabolic and transcriptomic analyses. The results showed dramatic and specific biochemical and transcriptomic changes in heat exposed berries, depending on the developmental stage and the stress duration. When applied at the herbaceous stage, HS delayed the onset of véraison. Heating also strongly altered the berry concentration of amino acids and organic acids (e.g. PHE, GABA, and malate) and decreased the anthocyanin content at maturity. These physiological alterations could be partly explained by the deep remodelling of transcriptome in heated berries. More than 7000 genes were deregulated in at least one of the nine experimental conditions. The most affected processes belong to the categories “stress responses”, “protein metabolism” and “secondary metabolisms”, highlighting the intrinsic capacity of grape berries to perceive heat stress and to build adaptive responses. Additionally, important changes in processes related to “transport”, “hormone” and “transcription” might contribute to the postponing of véraison. Finally, opposite effects depending on heating duration were observed for genes encoding enzymes of the general phenylpropanoid pathway, suggesting that the HS-induced decrease in anthocyanin content may result from a combination of transcript abundance and product degradation.

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Application of HPLC-PDA-MS metabolite profiling to investigate the effect of growth temperature and day length on blackcurrant fruit

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