Nyctanthes arbor-tristis is a nocturnal plant known for its intense floral scent. However, the reported molecular regulation of its volatile organic compound (VOC) biosynthesis remains enigmatic. In this study, we investigated the temporal floral VOCs analysis using GC-FID and GC-MS at five time points between 6 PM and 6 AM. Fifteen major VOCs were identified, including green leaf volatiles (GLVs), terpenoids, and phenylpropanoids/benzenoids (PBs). Among them, 4-ketoisophorone was the most abundant constituent at all time points, showing a peak concentration at midnight (18.8 ± 3.4%) and early morning (18.9 ± 4.5%) followed by benzaldehyde, with the highest abundance at 9 PM (12.7 ± 2.5%), and benzene acetaldehyde and methyl anthranilate, which showed a gradual increase during night and peaked in the morning hours. To uncover the transcriptional regulation underlying these temporal changes, RNA-Seq was performed on flowers collected at 6 PM, 9 PM, and 12 AM. A total of 25,913 high-confidence transcripts were annotated, and 68 differentially expressed genes (DEGs) were found to be common across comparisons. GO and KEGG enrichment revealed significant involvement of genes in secondary metabolism, circadian rhythm, hormone signaling, and environmental adaptation. Expression patterns of genes associated with GLV, benzenoid, and terpenoid biosynthesis were consistent with VOC emission trends. qRT-PCR analysis of selected structural genes and transcription factors further provided an independent experimental mechanism for pathway-specific transcriptional modulation across nocturnal stages. Together, these findings suggest that floral emission in N. arbor-tristis is governed by time-dependent coordination between metabolite accumulation and gene expression, providing insights into the molecular basis of nocturnal scent dynamics.
