Both Chuanxiong (Ligusticum chuanxiong Hort) and Tianma (Gastrodia elata Blume) have the effects of vasorelaxation and antihypertension. However, the effects of Tianshu Capsule (TSC, composed of Chuanxiong and Tianma in the mass ratio of 4:1) on antihypertensive activity have not been explored. This study aimed to investigate the eff ;ects of TSC on vascular tension and blood pressure in rats and to explore the underlying mechanisms. The vasorelaxant effect of TSC was explored on thoracic aortic rings (both intact endothelium and denuded) preincubated with phenylephrine (Phe) or potassium chloride (KCL). The mechanism was investigated in the presence of antagonists or blockers on aorta isolated from normotensive rats. The in vivo antihypertensive effect was assessed using a tail-cuff method on spontaneously hypertensive rats (SHRs). TSC (0.125-4 mg/mL) produced a concentration-dependent vasorelaxation on aortic rings preincubated with Phe (1 μM) or KCL (60 mM). Removal of aorta endothelium markedly attenuated the TSC activity. Pretreatment of aortic rings with β-adrenoceptor blocker propranolol (1 μM), muscarinic receptor antagonist atropine (1 μM), cyclooxygenase inhibitor indomethacin (IDO, 1 μM), adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536, 100 μM), K+ channel blockers 4-aminopyridine(4-AP, 1 mM) or barium chloride(BaCl2, 1 mM) followed by addition of Phe (1 μM) prior to TSC did not influence the TSC-induced relaxation. In contrast, the vasorelaxant effects of TSC were markedly inhibited by the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME, 10 μM), guanylyl cyclase inhibitor 1H- [1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 μM), K+ channel blockers, glibenclamide (100 μM) and clotrimazole (5 mM). Moreover, TSC (2 mg/mL, 4 mg/mL) inhibited CaCl2-induced contractions and caused a concentration-dependent rightward shift of the response curves. Additionally, TSC (2 mg/mL, 4 mg/mL) depressed the constriction caused by Phe (1 μM) in the absence of extracellular Ca2+. Furthermore, TSC (2.15 g/kg) lowered the systolic blood pressure (SBP), with no alteration in heart rate (HR) in SHRs. These findings demonstrated that TSC induced vasorelaxant effects via both endothelium-dependent and endothelium-independent pathways. The NO/sGC/cGMP pathway, ATP-sensitive K+ channels, Ca2+-activated K+ channels, inhibition of extracellular Ca2+ influx and intracellular Ca2+ release were probably involved in this relaxation. The vasorelaxant effects of TSC may make the greatest contribution to the reduction in high blood pressure.

As a group of the most important phytohormone, auxin homeostasis is regulated in a complex manner. Generally, auxin conjugations especially IAA glucosides are dominant on high auxin level conditions. Former terminal glucosylation researches mainly focus on O -position, while IAA- N -glucoside or IAA-Asp- N -glucoside has been neglected since their found in 2001. In our study, IAA-Asp- N -glucoside was firstly found specifically abundant (as high as 4.13 mg/g) in ginkgo seeds of 58 cultivars from Ginkgo Resource Nursery built in 1990. Furthermore, a novel N -glucosyltransferase GbNGT1, which could catalyze IAA-Asp and IAA to form their corresponding N -glucoside, was identified through differential transcriptome analysis and in vitro enzymatic test. The enzyme was demonstrated to possess specific catalyze capacity toward the N -position of IAA-amino acid or IAA among 52 substrates, and was typical of acid tolerance, metal ion independence and high temperature sensitivity. Docking and site-directed mutagenesis of this enzyme confirmed that E15G mutant could almost abolish enzyme catalytic activity towards IAA-Asp and IAA in vitro and in vivo . The IAA modification of GbNGT1 and GbGH3.5 was verified by transient expression assay in Nicotiana benthamiana . In conclusion, our results complement the terminal metabolic pathway of auxin, and the specific catalytic function of GbNGT1 towards IAA-amino acid provide a new way to biosynthesis indole-amide compounds. Highlight: The N-glucosylation of IAA or IAA-amino acids in auxin metabolism had been neglected over decades, our work for GbNGT1 redeems the missing chain of auxin metabolic pathway.

Gymnosperms represent an ancient lineage with diverse chemical profiles. Their terpenoids constitute a major class notable for substantial structural diversity. Terpene synthases (TPSs) are the driving force of terpene diversity. TPS-d is a gymnosperm-specific TPS subfamily, yet the evolution and functional diversity of TPS-d are still understudied. In this review, we analyze 134 functionally characterized gymnosperm TPSs to elucidate the evolutionary history and functional diversity of the predominant TPS-d subfamily. Phylogenetic analysis revealed substantial lineage-specific expansion and divergence within the TPS-d subfamily post-speciation. Crucially, we highlight the shifts in domain architecture during TPS-d evolution, providing structural evidence for the progressive attrition and eventual loss of the ancestral γ domain in TPS-d2 and TPS-d1 clades. Furthermore, we summarize the functional diversity of TPS-d enzymes in specialized metabolites, emphasizing their ecological roles in plant defense. Overall, this review provides a comprehensive overview of the evolutionary and functional landscape of the gymnosperm-specific TPS-d subfamily, serving as a valuable reference for future studies on TPSs in gymnosperms.