AUTHOR=Istiandari Pramesti , Yasumoto Shuhei , Seki Hikaru , Fukushima Ery Odette , Muranaka Toshiya 

TITLE=Class I and II NADPH-cytochrome P450 reductases exhibit different roles in triterpenoid biosynthesis in Lotus japonicus

JOURNAL=Frontiers in Plant Science

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2023.1214602

DOI=10.3389/fpls.2023.1214602

ISSN=1664-462X

ABSTRACT=<p>Cytochrome P450 monooxygenases (CYPs) are enzymes that play critical roles in the structural diversification of triterpenoids. To perform site-specific oxidations of the triterpene scaffold, CYPs require electrons transferred by NADPH-cytochrome P450 reductase (CPR), which is classified into two main classes, class I and class II, based on their structural difference. <italic>Lotus japonicus</italic> is a triterpenoids-producing model legume with one CPR class I gene (<italic>LjCPR1</italic>) and a minimum of two CPR class II genes (<italic>LjCPR2-1</italic> and <italic>LjCPR2-2</italic>). CPR classes I and II from different plants have been reported to be involved in different metabolic pathways. By performing gene expression analyses of <italic>L. japonicus</italic> hairy root culture treated with methyl jasmonate (MeJA), this study revealed that <italic>LjCPR1, CYP716A51</italic>, and <italic>LUS</italic> were down-regulated which resulted in no change in betulinic acid and lupeol content. In contrast, <italic>LjCPR2s, bAS, CYP93E1</italic>, and <italic>CYP72A61</italic> were significantly upregulated by MeJA treatment, followed by a significant increase of the precursors for soyasaponins, i.e. β-amyrin, 24-OH β-amyrin, and sophoradiol content. Triterpenoids profile analysis of <italic>LORE1</italic> insertion and hairy root mutants showed that the loss of the <italic>Ljcpr2-1</italic> gene significantly reduced soyasaponins precursors but not in <italic>Ljcpr1</italic> mutants. However, <italic>Ljcpr1</italic> and <italic>Ljcpr2-1</italic> mutants showed a significant reduction in lupeol and oleanolic, ursolic, and betulinic acid contents. Furthermore, <italic>LjCPR1</italic>, but not <italic>LjCPR2</italic>, was crucial for seed development, supporting the previous notion that CPR class I might support plant basal metabolism. This study suggests that CPR classes I and II play different roles in <italic>L. japonicus</italic> triterpenoid biosynthesis.</p>