# Triterpennoids Biosynthesis Regulation for Leaf Coloring of Wheel Wingnut (_Cyclocarya paliurus_)
## 4 Discussion  
Triterpenoids participate in plant communication, defense, and sensory control, and also have biological activities such as anti-inflammatory, anti-cancer, anti-bacterial, and antiviral [9]. _C. paliurus_ triterpenoid was found to be antidiabetic by promoting glucose uptake and improving lipid metabolism [24; 25; 26]. Furthermore, cyclocaric acid A, cyclocaric acid B, and cyclocarioside I, II, and III were ingredients particularly in _C. paliurus_, and cyclocarioside I sweetness is about 200 times that of sucrose [4]. Other triterpenoids including oleanolic acid and unsolic acid were also found to show anti-cancer bioactivity in animal and cell experiments [27]. _C. paliurus_ triterpenoid has been studied in previous research, including pentacyclic triterpenoid and secodammarane tr triterpenoid. Among them, ursanes-type, oleanane-type, and lupane-type belong to pentacyclic triterpenoid. Oleanane-type triterpenoid saponins have been identified in _C. paliurus_ leaves including oleanolic acid, arjunic acid, cyclocaric acid A and B; ursanes-type triterpenoid saponins have been identified including 2\(\alpha\)-Hydroxyursolic acid, 11-keto-ursolic acid, and asiatic acid; lupane-type triterpenoid saponins have been identified including betulinic acid [28]. At the same time, more dammarane-type triterpenoid saponins have been identified in recent years including cyclocarioside I, II, and III, and other dammarane-type triterpenoid saponins with unique structures that were uncovered gradually, including cyclocarioside O, cyclocarioside P, and cyclocarioside O-Q [29; 30]. In this study, the triterpenoids in LC1 leaf samples from different growing periods were measured by HPLC-MS. Madasitic acid, pomolic acid, 2\(\alpha\)-hydroxyusolic acid (ursanes-type triterpenoid), and 3-O-(2-O-acetyl-glucoseyl) oleanolic acid (oleanane-type triterpenoid) almost kept increasing during LC1 leaf development from red to green. [PERSON] et al. (2020) [31] considered that nitrogen forms alter total triterpenoid and triterpenoid accumulation simultaneously. It could be inferred that LC1 leaf development may lead to tissue nitrogen content variation leading to changes in the corresponding triterpenoids. Research related to _C. paliurus_ transcriptome and metabolome profiling is relatively limited. [PERSON] et al. (2020) [31] extracted RNA and used quantitative real-time polymerase chain reaction (qRT-PCR) analysis to study the triterpenoids accumulation in _C. paliurus_ clones, finding that HMGR, DXR, GPS, SQS, \(\beta\)-AS, and UDP were influenced by nitrogen forms and the trend was general. [PERSON] et al. (2020) [6] also found that the triterpenoid individuals of _C. paliurus_ germplasm exhibited the same geographical variation characteristics as total triterpenoids. In this study, the results are in agreement with [PERSON] et al. (2020) [31] and [PERSON] et al. (2020) [6] that the triterpenoid individuals nearly kept the same variation pattern, implying that the main gene regulation occurred upstream of the biosynthesis pathways. HMGS, HMGR, and FPPS showed significance during LC1 leaf development, and HMGS and HMGR belong to the MVA pathway, certifating our hypothesis. [PERSON] et al. (2021) cloned CpHMGR, CpDXR, and CpSQS genes and tried to uncover the gene expression patterns, finding that CpSQS exhibited higher gene expression in leaves than shoot from July to October while CpHMGR and CpDXR were expressed oppositely [5]. Partly in agreement with [PERSON] et al. (2021) [5], the downstream pathway genes FPPS in this study were found to play an important role in triterpen accumulation, while the upstream genes HMGS and HMGR in the MVA pathway also exhibited high expression. Other research on triterpenoids accumulation in different plant organs has been carried out including _Gleditsia sinensis_ [PERSON], _Clinopodium chinese_ ([PERSON].) [PERSON], and _Entada phaseoloides_ ([PERSON]) [PERSON]. [11; 12; 13], and the structure gene expression patterns between species were different.