Melatonin improves saponin biosynthesis and primary root growth in Psammosilene tunicoides hairy roots through multiple hormonal signaling and transcriptional pathways

Elicitation represents one of the most efficient techniques for improving metabolite biosynthesis and biomass accumulation in plant cell cultures. Melatonin (MLT) has been extensively studied for its multiregulatory functions, but its elicitation roles in medicinal plants remain largely unknown. Herein, we investigated the effects of MLT on triterpenoid saponin biosynthesis and root growth in hairy root cultures of Psammosilene tunicoides, an endangered Chinese medicinal plant famous for its pain-relieving capabilities. Exogenous MLT significantly boosted saponin accumulation, promoted primary root thickness and elongation, and increased biomass production compared with the control in P. tunicoides hairy roots, particularly when 10 mg L-1 MLT was applied for two days. Furthermore, the reactive oxygen species (ROS), nitric oxide (NO), and Ca2+ contents, as well as antioxidant enzyme activities, were initially triggered after MLT treatments. Additionally, MLT raised the diverse phytohormone profiles that were related to plant secondary metabolism (salicylic acid, jasmonic acid, abscisic acid) and growth (indoleacetic acid (IAA) conjugates, cytokinins, and strigolactone) but decreased free IAA levels in hairy roots. Analysis of comparative transcriptome data revealed that multiple differentially expressed genes (DEGs) might be the key regulators in MLT-elicited networks, especially genes that were associated with signal transduction (POD, NR, and CDPK), transcription factor regulation (WRKYs, NACs, and AR2/ERFs), terpenoid metabolism (HMGS, DXS, GPPS, and SE) and root growth (COBL7 and Egase). These findings demonstrated that MLT elicitors could simultaneously improve saponin biosynthesis and primary root growth in P. tunicoides hairy roots, mainly by enhancing the ROS-mediated signaling cascade and coregulating the associated phytohormonal and transcriptional pathways.