Effects of light irradiation on essential oil biosynthesis in the medicinal plant Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim) Kitag

Abstract

Asarum heterotropoides Fr. var. mandshuricum (Maxim) Kitag (Chinese wild ginger) is an important medicinal herb. Essential oil extracted from its roots is the key ingredient and is mainly composed of phenylpropanoid compounds. As a skiophyte plant, light is a crucial factor for A. heterotropoides var. mandshuricum growth and metabolism. To investigate the effects of light irradiation on the essential oil biosynthesis in A. heterotropoides var. mandshuricum, the plants were cultivated in four light irradiation treatments (100, 50, 24 and 12% full sunlight). The photosynthetic capacity, essential oil content and composition, activities of several enzymes and levels of some secondary metabolites involved in the shikimic acid and cinnamic acid pathways were analyzed. The leaf mass per area, average diurnal net photosynthetic rate, and the essential oil content increased significantly with increasing light intensity. Phenylalanine, cinnamic acid, and p-coumaric acid in the cinnamic acid pathway were at their highest levels in plants cultivated in 100% full sunlight. The highest content of shikimic acid in the shikimic acid pathway was obtained in plants grown in 50% sunlight transmittance. The activity of the enzymes 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase, phenylalanine ammonia lyase, cinnamate-4-hydroxylase and 4-coumarate:CoA ligase increased proportionally with light intensity. Overall, we conclude that high light irradiation promotes high net photosynthetic rate, high activity of enzymes and high amounts of phenylpropanoid precursor metabolites leading to significant biosynthesis of essential oil in A. heterotropoides var. mandshuricum.

Fig 1. Phenylpropanoid biosynthesis through the shikimic acid and cinnamic acid pathways (referring to Maeda et al.

[7] and Rastogi et al. [10]). PEP: phosphoenol pyruvate; C4H: cinnamate-4-hydroxylase and 4CL: 4-coumarate:CoA ligase; CM: chorismate mutase; PAL: phenylalanine ammonia lyase; DAHPS: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase; E4P: erythrose-4-phosphate.

Fig 2. Morphological and physiological responses to light treatments in A. heterotropoides var. mandshuricum.

The leaf mass per area (LMA) (A) and average diurnal net photosynthetic rate (Pn) (B) at three phenological stages (18 May, 25 May, 2 June) of A. heterotropoides var. mandshuricum grown in four light radiations. Note: I, 100% full sunlight; II, 50% full sunlight; III, 24% full sunlight; IV, 12% full sunlight. Error bar represents standard deviation (n = 5); different letters on the bars mean significant difference (P <0.05).

Fig 3. Essential oil content in fibrous roots of A. heterotropoides var. mandshuricum plants grown in different light irradiations.

Note: I, 100% full sunlight; II, 50% full sunlight; III, 24% full sunlight; IV, 12% full sunlight. Error bar represents standard deviation (n = 5); different letters on the bars mean significant difference (P <0.05).

Fig 4. Gas chromatogram of phenylpropanoid and aromatic compounds in essential oil from fibrous roots of A. heterotropoides var. mandshuricum plants grown in four light conditions.

A, 100% full sunlight; B, 50% full sunlight; C, 24% full sunlight; D, 12% full sunlight. cps = count per second.

Fig 5. Heatmap displaying the variation of the relative content of phenylpropanoid and aromatic compounds detected in the extracted essential oil from fibrous roots of A. heterotropoides var. mandshuricum grown in four light conditions.

Plot colors reflect the proportion of the detected metabolites within the essential oil, ranging from low (black) to high (red). Black means not detected. Treatment I, 100% full sunlight; Treatment II, 50% full sunlight; Treatment III, 24% full sunlight; Treatment IV, 12% full sunlight.

Fig 6. Activity key enzymes involved in the shikimic acid and cinnamic acid pathways.

Activity of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) (A), phenylalanine ammonia lyase (PAL) (B), cinnamate-4-hydroxylase (C4H) (C), 4-coumarate:CoA ligase (4CL) (D) in different tissues of A. heterotropoides var. mandshuricum grown in four light irradiations. I, 100% full sunlight; II, 50% full sunlight; III, 24% full sunlight; IV, 12% full sunlight. Error bar represents standard deviation (n = 5); different letters on the bars mean significant difference (P <0.05).

Fig 7. The ion chromatograms of four compounds.

(A) chromatogram of shikimic acid standard, (A₁) chromatogram of shikimic acid in A. heterotropoides var. mandshuricum sample; (B) chromatogram of phenylalanine standard, (B₁), chromatogram of phenylalanine in A. heterotropoides var. mandshuricum sample; (C), chromatogram of cinnamic acid standard, (C₁), chromatogram of cinnamic acid in A. heterotropoides var. mandshuricum sample; (D), chromatogram of p-coumaric acid standard, (D₁) chromatogram of p-coumaric acid in A. heterotropoides var. mandshuricum sample.

Fig 8. Effect of light treatments of four precursor metabolites involved in the shikimic acid and cinnamic acid pathways.

Shikimic acid (A), phenylalanine (B), cinnamic acid (C) and p-coumaric acid (D) contents in root, leaf and whole A. heterotropoides var. mandshuricum grown in different light irradiations. Note: I, 100% full sunlight; II, 50% full sunlight; III, 24% full sunlight; IV, 12% full sunlight. Error bar represents standard deviation (n = 5); different letters on the bars mean significant difference (P <0.05).