Flavonoid Accumulation Varies in Medicago truncatula in Response to Mercury Stress

Abstract

Mercury (Hg) contamination is increasing worldwide in both wild ecosystems and agricultural soils due to natural processes, but mostly to anthropic activities. The molecular mechanisms involved in Hg toxicity and tolerance in plants have been extensively studied; however, the role of flavonoids in response to Hg stress remains to be investigated. We conducted a metabolomic study to analyze the changes induced at the secondary metabolite level in three Hg-tolerant and one Hg-sensitive Medicago truncatula cultivars. A total of 46 flavonoid compounds, classified into five different flavonoid families: anthocyanidins, flavones, isoflavones, pterocarpan flavonoids, and flavanones, along with their respective glycoconjugate derivatives, were identified in leaf and root tissues. The synthesis of free isoflavones, followed by monoglycosylation and further malonylation was shown to be characteristic of root samples, whereas higher glycosylation, followed by further acylation with coumaric and ferulic acid was characteristic of leaf tissues. While minor changes were observed in leaves, significant quantitative changes could be observed in roots upon Hg treatment. Some flavonoids were strongly upregulated in roots, including malonylglucosides of biochanin A, formononetin and medicarpin, and aglycones biochanin, daidzein, and irisolidone. Hg tolerance appeared to be mainly associated to the accumulation of formononetin MalGlc, tricin GlcAGlcA, and afrormosin Glc II in leaves, whereas aglycone accumulation was associated with tolerance to Hg stress in roots. The results evidence the alteration of the flavonoid metabolic profile and their glycosylation processes in response to Hg stress. However, notable differences existed between varieties, both in the basal metabolic profile and in the response to treatment with Hg. Overall, we observed an increase in flavonoid production in response to Hg stress, and Hg tolerance appeared to be associated to a characteristic glycosylation pattern in roots, associated with the accumulation of aglycones and monoglycosylated flavonoids. The findings are discussed in the context of the flavonoid biosynthetic pathway to provide a better understanding of the role of these secondary metabolites in the response and tolerance to Hg stress in M. truncatula.

Keywords: LC-QTOF; Medicago truncatula; flavonoids; heavy metal stress; mercury; metabolomics.

FIGURE 1

High-resolution tandem mass spectrometry spectra obtained in the ESI(+)-QTOF-MS/MS mode for the identification of flavone glycoconjugates: (A) apigenin diglucuronide, (B) apigenin feruloyl triglucuronide, and (C) apigenin coumaroyl triglucuronide.

FIGURE 2

(A) Accumulation of flavonoid families in Medicago truncatula leaves and roots. (B) Dendrogram and heatmap of flavonoid families in M. truncatula leaves (L) and roots (R).

FIGURE 3

Relative abundance (%) of flavonoid families in Medicago truncatula leaves and roots.

FIGURE 4

Dendrogram and heatmap of the flavonoids and their glycoconjugates in Medicago truncatula leaves (-L) and roots (-R).

FIGURE 5

Flavonoid glycosylation profile in leaves and roots of Medicago truncatula. (A) Control samples, (B) Control vs Hg-treated varieties of leaves, and (C) Control vs Hg-treated varieties of roots. Agly, aglicone; GlcA, glucuronic acid; Glc, glucose; Mal, malonic acid; Fer, ferrulic acid; Cou, Coumaric acid; Gly, monoglycosylated; GlyGly, diglycosylated; GlyGlyGly, triglycosylated; and Acy, acylated.

FIGURE 6

Dendrogram and heatmap analysis of flavonoids and their glycoconjugates profiling in Medicago truncatula leaves (-L) and roots (-R) for control (C) and Hg-treated plants.

FIGURE 7

PCA score plots of control (C, blue) and Hg-treated varieties (Hg, red) of Medicago truncatula, based on individual flavonoid accumulation. (A) Leaves and (B) Roots.

FIGURE 8

PCA bi-plots of control (C, blue) and Hg-treated varieties (Hg, red) of Medicago truncatula, based on the flavonoids glycosylation degree. (A) Leaves and (B) Roots. Agly, aglycone; Gly, monoglycosylated; GlyGly, diglycosylated; GlyGlyGly, triglycosylated; and Acy, acylated.

FIGURE 9

Schematic diagram of the flavonoid biosynthesis pathway integrating the metabolomics results of differentially accumulated flavonoids upon Hg stress in Medicago truncatula leaf and root samples. Differentially expressed flavonoids are displayed in a color code based on the Log₂FC value; downregulated flavonoids (Log₂FC < 1.33) are indicated in green and upregulated components (Log₂FC > 1.33) are shown in red. CHI, chalcone isomerase; IFS, isoflavone synthase; F3H, flavanone 3 β-hydroxylase; 2HID, 2-hydroxyisoflavanone dehydratase; DFR, dihydroflavonol-4-reducatse; FNSII, flavone synthase; ANS, anthocyanidin synthase; I2’H, isoflavone 2’-hydroxylase; IFR, isoflavone reductase; VR, vestitone reductase; and DMID, dihydroxy-40-methoxy-isoflavanol dehydratase.