Rhododendron, a globally popular ornamental flower, is nevertheless limited in our understanding of the mechanisms underlying its fragrance formation. Notably, terpenoids are the most prevalent volatile metabolite produced by plants. In this study, gas chromatography–mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC–MS) and transcriptomics sequencing were conducted to analyze the synthesis mechanisms of terpenoid fragrance compounds of petals in fragrant R. fortunei Lindl. (YJ) and non-fragrant R. “Nova Zembla” (NW). The results identified that (-)-myrtenol, linalool, pinene, myrtenyl acetate, and terpineol were key floral aroma substances in YJ. Furthermore, an analysis of KEGG enrichment and differentially expressed genes (DEGs) revealed that the bud and decay stages exhibited the highest number of enriched DEGs among different aroma types, indicating these as critical stages for the synthesis of terpenoid floral compounds. In this study, a structural gene, denoted as RfFDPS, was identified as a negative regulatory gene for monoterpene accumulation and a positive regulatory gene for sesquiterpene accumulation in YJ. Utilizing subcellular localization technology, we determined that RfFDPS proteins are located in the cytoplasm. A functional analysis through transient expression and gene silencing of RfFDPS demonstrated its ability to regulate the accumulation of monoterpenes and sesquiterpenes. The overexpression of RfFDPS led to an increase in the expression of structural genes related to terpenoid synthesis, resulting in a decrease in monoterpenes and an increase in sesquiterpenes. Conversely, gene silencing had the opposite effect. In conclusion, RfFDPS plays a pivotal role in the synthesis and release of terpenoid volatile compounds in YJ petals, laying a solid theoretical foundation for the cultivation and enhancement of aromatic R. species.

Plant growth regulators Gibberellin A3 (GA₃) and N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) are widely used in ‘Shine Muscat’ cultivation to regulate berry shape and size. However, the molecular mechanisms underlying their regulation of berry shape remain poorly understood. This study was designed to elucidate the cytological processes and molecular basis through which GA₃ and CPPU modulate berry morphology in ‘Shine Muscat’. The results showed that spraying GA₃ or CPPU alone increases the hormone levels of endogenous auxin (IAA) and GA₃ and reduces the levels of endogenous 6-benzyladenine (6-BA). GA₃ treatment resulted in the number of cells per unit area being significantly reduced and the cell transverse and longitudinal diameters being significantly increased. CPPU treatment increases the number of cells per unit area, cell transverse and longitudinal diameters. In the results of CKvsG2 and CKvsC2 transcriptome sequencing, 2793 and 1082 differentially expressed genes (DEGs) were identified, respectively. These DEGs are significantly enriched in Gene Ontology (GO) terms related to plant hormones; the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the zeatin biosynthesis pathway (ko03030) is significantly enriched. The Arabidopsis response regulator (ARR) is down-regulated in response to GA₃ application and up-regulated in response to CPPU application. Transient overexpression of VvARR (OE-VvARR) in ‘Shine Muscat’ berry increased the number of berry cells and cell transverse and longitudinal diameters. Furthermore, virus-induced gene silencing of VvARR (VIGS-VvARR) reduced the number of berry cells but increased cell transverse and longitudinal diameters. The OE-VvARR grape hormone levels of endogenous GA₃, 6-BA, and IAA were significantly increased. In VIGS-VvARR grape, the levels of endogenous IAA and 6-BA are significantly increased, but there is no significant difference in endogenous GA₃. These findings offer novel insights into the molecular mechanisms by which GA₃ and CPPU govern berry development, corroborating the hypothesis that VvARR acts as a pivotal regulator mediating the effects of these plant growth regulators on berry cell morphology and, consequently, berry shape.

Aroma is a crucial quality trait in ornamental flowers; however, the molecular mechanisms by which hormones regulate fragrance in Rhododendron remain poorly understood. In this study, Gibberellin (GA₃)-treated petals of Rhododendron fortunei Lindl were used as experimental materials to integrate volatile metabolomics with RNA-seq analysis, aiming to investigate aroma changes and their underlying molecular regulatory mechanisms. We cloned and characterized RfHMGR1, which encodes a key enzyme in the Mevalonate (MVA) pathway, and verified its function. Subcellular localization analysis showed that the Green Fluorescent Protein (GFP) signal of the RfHMGR1-GFP fusion protein was mainly distributed in the cytoplasm. Transient overexpression of RfHMGR1 in petals of two Rhododendron species (R. fortunei and Rhododendron hybrida) significantly increased the accumulation of the terpenoid linalool, whereas gene silencing reduced linalool accumulation. Furthermore, the purified recombinant RfHMGR1 protein exhibited HMGR-specific reductase activity in vitro. Our results confirmed that GA₃ regulates the terpenoid fragrance of R. fortunei by targeting the MVA pathway gene RfHMGR1. Collectively, these findings provide new insights into the fragrance regulation mechanisms in R. fortunei and identify molecular targets for breeding strategies aimed at improving floral scent.

Rhododendron fortunei Lindl is known for its unique aroma, but the molecular mechanism behind plant hormone-mediated aroma biosynthesis remains unclear. To explore how brassinosteroids (BRs) and methyl jasmonate (MeJA) regulate its aroma, this study analyzed R. fortunei petal samples via physiological assays, volatile metabolome analysis, and transcriptome sequencing. Physiologically, BR/MeJA significantly increased the superoxide dismutase (SOD) and catalase (CAT) activity and decreased the malondialdehyde (MDA) content. Metabolome analysis identified 1268 volatile organic compounds (VOCs), with 265/70 VOCs up-/downregulated in the BR group and 248/181 VOCs up-/downregulated in the MeJA group compared to the controls. Transcriptome sequencing identified 19,333 differentially expressed genes (DEGs), which were enriched in pathways such as terpenoid and polyketide metabolism. Multi-omics screening revealed the candidate gene RfCYP92C6, whose transient overexpression in Nicotiana benthamiana increased the terpenoid content 2.2-fold. These findings clarify the aroma regulation mechanism of BRs/MeJA in R. fortunei and support the improvement of its aroma traits via genetic engineering.