MetaboLights MTBLS773 - GNPS Application of HPLC-PDA-MS metabolite profiling to investigate the effect of growth temperature and day length on blackcurrant fruit

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.

引言：黑加仑（Ribes nigrum L.）是兼具健康应用价值的典型“超级水果”。现有研究表明，基因型与栽培环境均可影响黑加仑的化学成分组成，尤其是抗坏血酸与各类酚类物质。温度、太阳辐射、降水量等环境因子，同样会对果实的化学成分产生显著影响。而当前全球气候正经历且将持续发生可预测的变化，进一步凸显了本研究的重要意义。

研究目的：本研究旨在采用非靶向高效液相色谱-光电二极管阵列-质谱（high performance liquid chromatography–photo diode array–mass spectrometry, HPLC-PDA-MS）代谢组学方法，解析花后环境条件（即温度与日长）对黑加仑果实品质及化学成分的影响，以期为该领域提供全新认知与更深入的理解。

研究方法：本研究开展人工气候室栽培试验，以单茎盆栽黑加仑品种‘Narve Viking’为试材，设置12℃、18℃、24℃三个恒温梯度，以及三种不同光周期处理（短日照、夜间打断短日照、自然夏季日照条件），同时设置室外自然环境对照组。采用非靶向HPLC-PDA-MS代谢组学方法对成熟浆果进行分析，以检测受控气候因子影响下的分子物质种类与浓度。

研究结果：本研究的非靶向代谢组学数据集在电喷雾电离（electrospray ionisation, ESI）正、负两种电离模式下，共获得7274个去卷积保留时间-质荷比（m/z）对；基于精确质量质谱分析，从中鉴定或初步注释得到549种代谢物。首先采用常规主成分分析（principal component analysis, PCA）结合弗里德曼显著性检验，筛选出对温度呈线性响应的代谢物；随后采用多块分层主成分分析结合弗里德曼显著性检验，筛选出对不同日长条件产生响应的代谢物。结果显示，温度对共计365种涵盖不同化学类别的代谢物具有显著影响。与受控环境条件下的果实相比，自然环境条件下成熟的黑加仑浆果中，34种已注释代谢物（主要为花青素与黄酮类物质）的积累量显著升高。在不同日长条件下，共有18种代谢物呈现差异调控模式。此外，针对丰度最高的花青素进行靶向与非靶向分析对比，结果显示两种分析方法具有高度一致性。本研究不仅证实了非靶向代谢组学方法的应用价值——可检测到传统靶向分析极易遗漏的大量种类丰富、丰度变化显著的代谢物，同时也证明了非靶向分析方法相较于靶向分析的精准性与可靠性。

研究结论：在受控自然环境条件下的黑加仑成熟过程中，本研究揭示了诸多环境因子与果实化学成分间的深层关联。在最高温度处理组中，丰度最高的花青素出现显著降低，这表明在极端炎热的夏季年份，黑加仑浆果的总花青素积累量普遍会有所下降。HPLC-PDA-MS代谢组学是分析富含酚类物质的浆果化学成分的优质方法。此外，本研究通过可控人工气候室试验，进一步加深了对植物与环境间相互作用的认知。