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.