Abstract
Introduction: Bird strikes are one of the greatest threats to aviation safety in the worldwide. Sphagneticola trilobata (L.) Pruski has anti-insect effects that may indirectly decrease bird populations, thereby reducing bird strikes. However, it is unclear how S. trilobata exerts its anti-insect effects. Moreover, the mechanism mediating the biosynthesis of its main volatile compounds is unknown. Objectives: This study was conducted to identify the major anti-insect volatile compounds in S. trilobata and elucidate their biosynthetic mechanisms. Methods: A continuous sampling method was used to analyze the released volatiles of S. trilobata. Direct feeding or fumigation treatments with the main monoterpenes were followed by the evaluation of anti-insect functions. Evolutionary and enzyme activity analyses were performed to verify the functions of target enzymes. The subcellular localization and potential functions of the target enzymes were revealed by quantitative analyses of synthase gene expression, subcellular localization experiments, inhibitor experiments, and enzyme activity analysis of proteins from different subcellular organelles. Results: α-Phellandrene, limonene, and p-cymene, which had a circadian release pattern, were the major volatiles in S. trilobata. These three monoterpenes have anti-insect functions. Additionally, StTPS3 has a relatively broad substrate specificity in vitro, which may result in the production of limonene, p-cymene, and β-caryophyllene. The circadian rhythm in StTPS3 expression was consistent with the changes in volatile compound levels. The encoded enzyme was localized in the cytoplasm. Inhibition of the mevalonate pathway reduced monoterpene formation. Proteins extracted from the cytoplasm and chloroplasts may catalyze the synthesis and conversion of monoterpene precursors. Conclusion: The study data provide direct evidence for the anti-insect effects of S. trilobata, while positively elucidating the biosynthesis of key volatiles from cytoplasmic GPP and NPP. Furthermore, the findings may be relevant for the control of bird populations at airports and the reduction of the risk of bird strikes.