Medicago truncatula CYP716A12 is a multifunctional oxidase involved in the biosynthesis of hemolytic saponins

Abstract

Saponins, a group of glycosidic compounds present in several plant species, have aglycone moieties that are formed using triterpenoid or steroidal skeletons. In spite of their importance as antimicrobial compounds and their possible benefits for human health, knowledge of the genetic control of saponin biosynthesis is still poorly understood. In the Medicago genus, the hemolytic activity of saponins is related to the nature of their aglycone moieties. We have identified a cytochrome P450 gene (CYP716A12) involved in saponin synthesis in Medicago truncatula using a combined genetic and biochemical approach. Genetic loss-of-function analysis and complementation studies showed that CYP716A12 is responsible for an early step in the saponin biosynthetic pathway. Mutants in CYP716A12 were unable to produce hemolytic saponins and only synthetized soyasaponins, and were thus named lacking hemolytic activity (lha). In vitro enzymatic activity assays indicate that CYP716A12 catalyzes the oxidation of β-amyrin and erythrodiol at the C-28 position, yielding oleanolic acid. Transcriptome changes in the lha mutant showed a modulation in the main steps of triterpenic saponin biosynthetic pathway: squalene cyclization, β-amyrin oxidation, and glycosylation. The analysis of CYP716A12 expression in planta is reported together with the sapogenin content in different tissues and stages. This article provides evidence for CYP716A12 being a key gene in hemolytic saponin biosynthesis.

Figure 1.

Comparison between lha-1 Mutant Line and Control Line. (A) Microhemolytic test on a blood agar plate: Black arrows show the absence of hemolysis in lha-1 mutant; white arrows show the positive controls; the spots without indications are extracts of lines from the activation tagging collection. (B) TLC analysis of purified saponins from control (1) and lha-1 mutant (2). (C) HPLC analysis of purified saponins from control (1) and lha-1 mutant (2).

Figure 2.

Analysis of Sapogenin Content in Leaves and Roots of lha-1 Mutant Line and Control Line. (A) GC-MS chromatograms of sapogenins in roots: lha-1 mutant (1) and control line (E113) (2). (B) and (C) Sapogenin content obtained by GC-FID analysis in leaves and roots, respectively; values for lha-1 (gray bars) and the control line (white bars) are means ± se of three biological replicates (three to 10 plants/replicate). Identification of soyasapogenol B was achieved considering all the artifactual compounds detected (soyasapogenols C, D, and F). *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 with F test). bayo, bayogenin; DM, dry matter; hed, hederagenin; IS, internal standard; med, medicagenic acid; olea, oleanolic acid; soya, soyasapogenol; zan, zanhic acid.

Figure 3.

Identification of the T-DNA–Tagged Locus and Expression of the CYP716A12-Tagged Gene in the lha-1 Mutant. (A) Location of the T-DNA insertion in CYP716A12 gene (not drawn to scale). The relative location and orientation of the T-DNA are shown. The T-DNA right border (RB) with four copies of the 35S enhancer was inserted at the end of the first exon producing the loss of small parts of the exon (in white) and the intron (hatched). LB, left border; UTR, untranslated region. (B) RT-PCR analysis of transcript level of CYP716A12 gene in leaves and roots of the control line (E113) and the lha-1 mutant. C-, negative control; Msc27, reference gene.

Figure 4.

Plants of lha-1 (E25-10) and Control Line (E113). Ten-week-old lha-1 mutant plant is reduced in growth compared with the contemporary control (E113) plant. Bar = 10 cm.

Figure 5.

Sapogenin Content in Mutant and Control Plants. Data were obtained by GC-FID analysis of lha-2 (CYP2768) and lha-3 (CYP3201) mutant lines (M3 generation) from a TILLING collection and the respective control plants. (A) lha-2: Mutant (gray bars, nine plants) and control Jemalong 2HA10-9-3 (black bars, three plants). (B) lha-3: Homozygous mutant (gray bars, three plants), heterozygous mutant (white bars, three plants), and wild type (black bars, three plants). Values are means ± se. (*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 with F test and linear contrasts). bayo, bayogenin; DM, dry matter; med, medicagenic acid; soya, soyasapogenol; spg, sapogenins; wt, wild type; zan, zanhic acid.

Figure 6.

In Vitro Oxidation of β-Amyrin and Erythrodiol by CYP716A12 in Microsomes of the WAT11 Strain. (A) GC-MS analysis of the reaction products resulting from in vitro assay containing β-amyrin (1 and 2) and erythrodiol (3 and 4) as substrate on strains expressing CYP716A12 (black) and control (gray). (B) Mass spectrum of the peaks at 26.75 min indicated by the arrows in (A): The retention time and mass spectra of these peaks compare well with those of oleanolic acid.

Figure 7.

Expression Analysis of CYP716A12 Gene in Control Line (E113) by Quantitative RT-PCR: Different Organs at Different Phenological Stages Were Considered. Data are shown for leaves (light gray bars), roots (dark gray bars), stems (white bars), pods (black bars), flowers (light cross-hatched bars), and nodules (dark cross-hatched bars). Values are means ± se of three biological replicates (three plants/replicate) and are expressed relative to leaves in the vegetative stage.

Figure 8.

Sapogenin Content Obtained by GC-FID Analysis in Control Line (E113): Different Organs at Different Phenological Stages Were Reported. Values are means ± se of three biological replicates (three plants/replicate). bayo, bayogenin; DM, dry matter; hed, hederagenin; med, medicagenic acid; olea, oleanolic acid; soya A, soyasapogenol A; soyaB, soyasapogenol B; zan, zanhic acid.

Figure 9.

Role of CYP716A12 in Sapogenin Biosynthesis. (A) Oxidation steps catalyzed by CYP716A12. (B) Hypothetical sapogenin biosynthetic pathway in M. truncatula.