Histochemical and molecular analyses reveal an insight into the scent volatiles synthesis and emission in ephemeral flowers of Murraya paniculata (L.) Jack.

Main conclusion: Temporal histolocalization of floral volatiles in the petal epidermis of Murraya paniculata was found to be linked with the coordinated expression of candidate genes and successive accumulation of an internal pool of volatiles. Murraya paniculata (Rutaceae) is known for its highly fragrant ephemeral flowers that emit volatiles to attract nocturnal pollinators. To unfold the patterns of volatile emission in relation to floral life-span, we studied time-course accumulation and emission rate of scent volatiles at six timepoints of floral maturation, at an interval of 4 h starting from the bud stage to the senescence stage on the next day. This study revealed the maximum emission rate of scent volatiles at the anthesis stage at 18:00 h. This finding correlates well with the maximum accumulation of volatiles in the internal pool of the flowers at this stage. The key volatiles detected in both emitted and internal pools were benzaldehyde, benzeneacetaldehyde, linalool, caryophyllene, germacrene-D and α-farnesene. In addition, the internal pool also contained substantial amounts of indole, scopoletin, caffeine and osthole. To histochemically localize the temporal accumulation of major volatile groups in the epidermal cells, petal cross sections were stained with NaDi and ferric chloride to visualize terpenes and phenolics, respectively, under light microscope. Histolocalization studies showed a higher accumulation of terpenes at 14:00 h and 18:00 h, which subsequently was reduced as senescence approached. Significant phenolics in the abaxial and adaxial layers of the petal epidermis accumulated at 18:00 h and at the early senescence (06:00 h) stages. Furthermore, temporal localization of active shikimate dehydrogenase (SKDH) protein through in-gel activity assay demonstrated higher enzymatic activities at anthesis (18:00 h) and fully bloomed (02:00 h) stages, supporting the findings of higher accumulation of phenolic volatiles at 18:00 h and 06:00 h stages. Expression analysis of major candidate genes of floral scent volatiles pathway supported the hypothesis that the emission rate of floral fragrance reached its maximum at the anthesis (18:00 h) stage. In contrast, biosynthesis of scent compounds started at the bud (14:00 h) stage itself as indicated by the RT-PCR semi-quantitative estimation. As flowers of M. paniculata attract multiple pollinator species, this study could also serve as a springboard for pollination biology in Rutaceae, which includes important fruit crops.