Transcriptomics Integrated with Free and Bound Terpenoid Aroma Profiling during “Shine Muscat” (Vitis labrusca × V. vinifera) Grape Berry Development Reveals Coordinate Regulation of MEP Pathway and Terpene Synthase Gene Expression

Terpenes and their derivatives are important biomarkers of grape quality as they contribute to the flavor and aroma of grapes. However, the molecular basis of terpene biosynthesis throughout the grapevine phenological developmental cycle remains elusive. Our current study investigates the free and bound terpene biosynthesis of berries at different phenological stages from preveraison to harvest. Detailed gene expression (transcriptomics) analysis, terpenoid volatile production by gas chromatography–mass spectrometry (GC–MS), and in planta transient expression were employed. Our results show that concentrations of most individual terpenes at different stages are distinctive and increase from preveraison to the veraison stage followed by a decrease from veraison to maturity. The combined transcriptomic analysis and terpene profiling revealed that 22 genes belonging to the MEP pathway and multiple classes of transcription factor family members including bHLH and several hormone biosynthesis- or signaling-related genes likely participate in the regulation of terpenoid biosynthesis according to their specific expression patterns in berries. Quantitative real-time polymerase chain expression analysis of 8 key differentially expressed genes in MEP pathways and further 12 randomly selected genes was performed during 8 sampling stages and validated the RNA-seq-derived expression profiles. To further confirm the function of a subset of the differentially expressed genes, we investigated the effects of combined overexpression of 1-deoxy-d-xylulose-5-phosphate synthase (VvDXS1-LOC100249323), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (VvDXR-LOC100248516), and terpene synthase (VvTPS56-LOC100266449) on the production of terpenes by transient overexpression in Nicotiana benthamiana leaves. The overall developmental patterns of total terpenes and gene expression profiles will help guide the functional analyses of further candidate genes important for terpene biosynthesis of grape as well as identifying the master transcriptional and hormonal regulators of this pathway in the future.萜类及其衍生物是葡萄品质的重要生物标志物，其对葡萄的风味与香气形成具有关键贡献。然而，贯穿葡萄物候发育周期的萜类生物合成分子机制仍未明晰。本研究针对葡萄浆果从转色前至采收期的不同物候阶段，探究其游离型与结合型萜类的生物合成过程。研究采用了基因表达（转录组学）深度分析、气相色谱-质谱联用（GC-MS）检测萜类挥发性产物，以及植物体内瞬时表达实验等技术手段。结果表明，多数单一萜类物质在不同物候阶段的含量存在显著差异：从转色前至转色期呈上升趋势，随后从转色期至成熟期逐渐下降。联合转录组分析与萜类谱分析发现，22个属于甲基赤藓醇磷酸（MEP）通路的基因、包括碱性螺旋-环-螺旋（bHLH）在内的多类转录因子家族成员，以及若干激素生物合成或信号转导相关基因，依据其在浆果中的特异性表达模式，推测可能参与萜类生物合成的调控。本研究在8个采样阶段中，对MEP通路中的8个关键差异表达基因以及额外12个随机选取的基因开展了实时定量聚合酶链反应分析，验证了RNA-seq得到的表达谱数据。为进一步验证部分差异表达基因的功能，本研究通过在本氏烟草（Nicotiana benthamiana）叶片中瞬时过表达1-脱氧-D-木酮糖-5-磷酸合酶（VvDXS1-LOC100249323）、1-脱氧-D-木酮糖-5-磷酸还原异构酶（VvDXR-LOC100248516）以及萜类合酶（VvTPS56-LOC100266449），探究了三者联合过表达对萜类物质生成的影响。总萜类物质的整体发育模式与基因表达谱，将为后续解析葡萄萜类生物合成相关候选基因的功能提供指导，同时也有助于未来鉴定该通路的核心转录与激素调控因子。