Comprehensive Quantification of (Poly)phenols in Lotus japonicus with and without Arbuscular Mycorrhizal Symbiosis

Abstract

In the present study, a highly specific, accurate, and robust ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous quantification of 50 plant (poly)phenol analytes was developed and validated to assess the effect of arbuscular mycorrhizal (AM) symbiosis on the (poly)phenolic content of the model legume Lotus japonicus (L. japonicus). Determination of molar concentrations of analytes in roots and shoots of wild-type and AM mutant L. japonicus (with and without AM symbiosis, respectively) revealed an overall increase in (poly)phenols in mycorrhizal plants. Time-course observation over 10 weeks showed a shift in (poly)phenol concentrations, especially in the roots. In total, 13 analytes were notably more abundant in young AM roots, suggesting a potential role in symbiosis initiation. An accumulation of various (poly)phenols at later stages of symbiosis might indicate a potential involvement in arbuscule degradation or AM autoregulation.

Keywords: Lotus japonicus; UHPLC−MS/MS; arbuscular mycorrhiza; flavonoids; plant polyphenols; quantification method; symbiosis.

1

Chemical structures of polyphenol analytes and internal standards (IS) from the ultrahigh-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UHPLC–ESI–MS/MS) method: catechin (1), epicatechin (2), genistin (3), demethylwedelolactone (4; IS), sophoricoside (5), myricetin (6), thevetiaflavone (7; IS), moracin M (8; IS), daidzein (9), 2′-hydroxygenistein (10), liquiritigenin (11), quercetin (12), luteolin (13; IS), calycosin (14), wedelolactone (15), coumestrol (16), genistein (17), apigenin (18), kaempferol (19), vestitone (20), naringenin (21), 2′-hydroxyformononetin (22), cladrin (23), syringetin (24), isorhamnetin (25), hesperetin (26), 7-hydroxy-5,4′-dimethoxyisoflavone (27), lupinalbin A (28), ayamenin D (29), mosloflavone (30), isoliquiritigenin (31), formononetin (32), vestitol (33), maackiain (34), lotuscarpene (35), medicarpin (36), genkwanin (37), acacetin (38), glyceollin I (39), biochanin A (40), phaseollin (41), lotuschromone (42), luteone (43), sativan (44), salvigenin (45), 7,3′,4′-trimethylluteolin (46), isosativan (47), lotusaldehyde (48), wighteone (49), glyurallin A (50; IS), 5-hydroxy-7,4′-dimethoxyflavone (51), licoisoflavone B (52), 3,9-dimethoxycoumestan (53), 5-hydroxy-7,4′-dimethoxyisoflavone (54), lupinalbin B (55), 3,7,4′-trimethylkaempferol (56; IS), and alpinumisoflavone (57; IS).

2

(A) Ultrahigh-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UHPLC–ESI–MS/MS) chromatogram showing quantifier mass transitions in polarity switching mode (ESI±) of distinct polyphenol analytes (n = 50) and internal standards (IS; n = 7). (B–O) Separation of isobaric analytes in a reference standard mixture. Most isobaric analytes are separated chromatographically except for (H) 1 (m/z 288.9 → 245.0) and 2 (m/z 288.9 → 108.9), (D) 17 (m/z 268.9 → 132.8) and 18 (m/z 268.9 → 116.9), (G) 19 (m/z 285.0 → 185.0) and 20 (m/z 284.9 → 108.9), and (L) 45 (m/z 329.0 → 268.0) and 46 (m/z 329.0 → 312.9), which exhibit distinct multiple reaction monitoring (MRM) transitions.

3

Normalized heat maps of analyte concentrations in wild-type (WT) and mutant (pooled allelic mutants of ccamk, cyclops, ram1, and ram2) Lotus japonicus harvested 7 and 10 weeks postinoculation (wpi). Mycorrhizal (AM) and nonmycorrhizal (control) (A) roots and (B) shoots are shown.

4

Temporal development of analyte concentrations in AM (red) and control (blue) roots of wild-type (WT) Lotus japonicus after 2, 4, 6, 8, and 10 weeks postinoculation (wpi), including confidence bands (gray).

5

Temporal development of analyte concentrations in AM (red) and control (blue) aboveground organs (shoots) of wild-type (WT) L. japonicus after 2, 4, 6, 8, and 10 weeks post inoculation (wpi), including confidence bands (gray).