Functional Characterization of Double-Bond Reductases in Dihydro-β-Ionone Biosynthesis in Cymbidium sinense

<i>Cymbidium sinense</i> is a highly valued ornamental orchid renowned for its strong floral fragrance. In this study, dihydro-β-ionone was identified as a major volatile compound in <i>C. sinense</i> 'Qi Hei'. Its emission increased progressively during flower development and was predominantly released from the sepals and petals. Transcriptome analysis of flowers at three developmental stages led to the identification of four double-bond reductase genes, designated <i>CsDBR1-CsDBR4</i>. Spatiotemporal expression profiling demonstrated that transcript levels of <i>CsDBRs</i> were highest in sepals and petals, showing a significant positive correlation with dihydro-β-ionone accumulation (<i>p</i> < 0.05). Heterologous expression in <i>Escherichia coli</i> and subsequent in vitro enzymatic assays confirmed that recombinant CsDBR1, CsDBR2, and CsDBR4 proteins catalyze the conversion of β-ionone to dihydro-β-ionone, whereas CsDBR3 exhibited no detectable activity. Transient expression in <i>Nicotiana benthamiana</i> leaves further verified the in planta function of <i>CsDBR1</i>, <i>CsDBR2</i>, and <i>CsDBR4</i>, resulting in elevated production of dihydro-β-ionone upon infiltration of β-ionone. Substrate specificity assays revealed that CsDBR2 and CsDBR4 also reduced 1-octen-3-one, 3-nonen-2-one, and pulegone. Collectively, these findings demonstrate that CsDBR1, CsDBR2, and CsDBR4 are key enzymes responsible for dihydro-β-ionone biosynthesis in <i>C. sinense</i>, providing a genetic foundation for molecular breeding aimed at improving floral fragrance in orchids.