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

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. Lotus japonicus is a triterpenoids-producing model legume with one CPR class I gene (LjCPR1) and a minimum of two CPR class II genes (LjCPR2-1 and LjCPR2-2). CPR classes I and II from different plants have been reported to be involved in different metabolic pathways. By performing gene expression analyses of L. japonicus hairy root culture treated with methyl jasmonate (MeJA), this study revealed that LjCPR1, CYP716A51, and LUS were down-regulated which resulted in no change in betulinic acid and lupeol content. In contrast, LjCPR2s, bAS, CYP93E1, and CYP72A61 were significantly upregulated by MeJA treatment, followed by a significant increase of the precursors for soyasaponins, i.e. ß-amyrin, 24-OH ß-amyrin, and sophoradiol content. Triterpenoids profile analysis of LORE1 insertion and hairy root mutants showed that the loss of the Ljcpr2-1 gene significantly reduced soyasaponins precursors but not in Ljcpr1 mutants. However, Ljcpr1 and Ljcpr2-1 mutants showed a significant reduction in lupeol and oleanolic, ursolic, and betulinic acid contents. Furthermore, LjCPR1, but not LjCPR2, 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 L. japonicus triterpenoid biosynthesis.

Lotus japonicus Triterpenoid Profile and Characterization of the CYP716A51 and LjCYP93E1 Genes Involved in Their Biosynthesis In Planta.

Lotus japonicus is an important model legume plant in several fields of research, such as secondary (specialized) metabolism and symbiotic nodulation. This plant accumulates triterpenoids; however, less information regarding its composition, content and biosynthesis is available compared with Medicago truncatula and Glycine max. In this study, we analyzed the triterpenoid content and composition of L. japonicus. Lotus japonicus accumulated C-28-oxidized triterpenoids (ursolic, betulinic and oleanolic acids) and soyasapogenols (soyasapogenol B, A and E) in a tissue-dependent manner. We identified an oxidosqualene cyclase (OSC) and two cytochrome P450 enzymes (P450s) involved in triterpenoid biosynthesis using a yeast heterologous expression system. OSC9 was the first enzyme derived from L. japonicus that showed α-amyrin (a precursor of ursolic acid)-producing activity. CYP716A51 showed triterpenoid C-28 oxidation activity. LjCYP93E1 converted ß-amyrin into 24-hydroxy-ß-amyrin, a metabolic intermediate of soyasapogenols. The involvement of the identified genes in triterpenoid biosynthesis in L. japonicus plants was evaluated by quantitative real-time PCR analysis. Furthermore, gene loss-of-function analysis of CYP716A51 and LjCYP93E1 was conducted. The cyp716a51-mutant L. japonicus hairy roots generated by the genome-editing technique produced no C-28 oxidized triterpenoids. Likewise, the complete abolition of soyasapogenols and soyasaponin I was observed in mutant plants harboring Lotus retrotransposon 1 (LORE1) in LjCYP93E1. These results indicate that the activities of these P450 enzymes are essential for triterpenoid biosynthesis in L. japonicus. This study increases our understanding of triterpenoid biosynthesis in leguminous plants and provides information that will facilitate further studies of the physiological functions of triterpenoids using L. japonicus.

Lotus japonicus Genetic, Mutant, and Germplasm Resources.

A quarter of a century has passed since Lotus japonicus was proposed as a model legume because of its suitability for molecular genetic studies. Since then, a comprehensive set of genetic resources and tools has been developed, including recombinant inbred lines, a collection of wild accessions, published mutant lines, a large collection of mutant lines tagged with LORE1 insertions, cDNA clones with expressed sequence tag (EST) information, genomic clones with end-sequence information, and a reference genome sequence. Resource centers in Japan and Denmark ensure easy access to data and materials, and the resources have greatly facilitated L. japonicus research, thereby contributing to the molecular understanding of characteristic legume features such as endosymbiosis. Here, we provide detailed instructions for L. japonicus cultivation and describe how to order materials and access data using the resource center websites. The comprehensive overview presented here will make L. japonicus more easily accessible as a model system, especially for research groups new to L. japonicus research. © 2018 by John Wiley & Sons, Inc.

User Guide for the LORE1 Insertion Mutant Resource.

Lotus japonicus is a model legume used in the study of plant-microbe interactions, especially in the field of biological nitrogen fixation due to its ability to enter into a symbiotic relationship with a soil bacterium, Mesorhizobium loti. The LORE1 mutant population is a valuable resource for reverse genetics in L. japonicus due to its non-transgenic nature, high tagging efficiency, and low copy count. Here, we outline a workflow for identifying, ordering, and establishing homozygous LORE1 mutant lines for a gene of interest, LjFls2, including protocols for growth and genotyping of a segregating LORE1 population.

The deubiquitinating enzyme AMSH1 is required for rhizobial infection and nodule organogenesis in Lotus japonicus.

Legume-rhizobium symbiosis contributes large quantities of fixed nitrogen to both agricultural and natural ecosystems. This global impact and the selective interaction between rhizobia and legumes culminating in development of functional root nodules have prompted detailed studies of the underlying mechanisms. We performed a screen for aberrant nodulation phenotypes using the Lotus japonicus LORE1 insertion mutant collection. Here, we describe the identification of amsh1 mutants that only develop small nodule primordia and display stunted shoot growth, and show that the aberrant nodulation phenotype caused by LORE1 insertions in the Amsh1 gene may be separated from the shoot phenotype. In amsh1 mutants, rhizobia initially became entrapped in infection threads with thickened cells walls. Some rhizobia were released into plant cells much later than observed for the wild-type; however, no typical symbiosome structures were formed. Furthermore, cytokinin treatment only very weakly induced nodule organogenesis in amsh1 mutants, suggesting that AMSH1 function is required downstream of cytokinin signaling. Biochemical analysis showed that AMSH1 is an active deubiquitinating enzyme, and that AMSH1 specifically cleaves K63-linked ubiquitin chains. Post-translational ubiquitination and deubiquitination processes involving the AMSH1 deubiquitinating enzyme are thus involved in both infection and organogenesis in Lotus japonicus.