Multi-omics analyses reveal microRNAs ' role in terpene biosynthesis regulation in slash pine

Slash pine ( Pinus elliottii ) is a crucial resin-tapping tree species in China, and its resin, an important product, is predominantly composed of monoterpenes and diterpenes. Although the influence of transcription factors (TFs) and microRNAs (miRNAs) on terpene biosynthesis is well known, the specific effect of miRNAs on terpene synthesis in slash pine remains insufficiently explored. This study employed comprehensive analyses, including transcriptome sequencing, small RNA sequencing, and degradation profiling, focusing on the bark and needles of slash pine at three distinct time points to unveil the miRNA-gene regulatory patterns governing terpene synthesis. The transcriptome analysis identified 19,673 differentially expressed genes (DEGs), including 726 TF-coding genes, 59 genes related to terpene synthesis, 66 ABC transporter genes, and 317 CYP450 genes. Small RNA sequencing identified 175 miRNAs, with 105 differentially expressed miRNAs (DEMs) targeting 2497 genes. Notably, pta-miR949, pta-miR946a-3p, and pab-miR3710 targeted PITA39236 (HDS), PITA24638 (GPPS), and PITA12789 (ACAT), respectively. Temporal analysis revealed that DEMs and DEGs in Profiles 7 and 13 significantly influenced terpene synthesis in slash pine. Specifically, bHLH genes (Novel9385, PITA08062, PITA23693, and PITA43216) in these expression profiles, as well as the pta-miR858a/b-PITA03784/PITA28059/PITA18794 (MYB) and pta-miR159c-Novel1571/Novel4202 (MYB) modules, play pivotal roles in slash pine terpene synthesis. This study, for the first time, revealed the regulatory role of miRNAs in slash pine terpene synthesis, providing a foundation for understanding the involvement of miRNAs in this process and offering theoretical support for future molecular breeding efforts aimed at enhancing resin yield in slash pine.