Manninotriose is a major carbohydrate in red deadnettle (Lamium purpureum, Lamiaceae)

Abstract

Background and aims: There is a great need to search for natural compounds with superior prebiotic, antioxidant and immunostimulatory properties for use in (food) applications. Raffinose family oligosaccharides (RFOs) show such properties. Moreover, they contribute to stress tolerance in plants, acting as putative membrane stabilizers, antioxidants and signalling agents.

Methods: A large-scale soluble carbohydrate screening was performed within the plant kingdom. An unknown compound accumulated to a high extent in early-spring red deadnettle (Lamium purpureum) but not in other RFO plants. The compound was purified and its structure was unravelled with NMR. Organs and organ parts of red deadnettle were carefully dissected and analysed for soluble sugars. Phloem sap content was analysed by a common EDTA-based method.

Key results: Early-spring red deadnettle stems and roots accumulate high concentrations of the reducing trisaccharide manninotriose (Galα1,6Galα1,6Glc), a derivative of the non-reducing RFO stachyose (Galα1,6Galα1,6Glcα1,2βFru). Detailed soluble carbohydrate analyses on dissected stem and leaf sections, together with phloem sap analyses, strongly suggest that stachyose is the main transport compound, but extensive hydrolysis of stachyose to manninotriose seems to occur along the transport path. Based on the specificities of the observed carbohydrate dynamics, the putative physiological roles of manninotriose in red deadnettle are discussed.

Conclusions: It is demonstrated for the first time that manninotriose is a novel and important player in the RFO metabolism of red dead deadnettle. It is proposed that manninotriose represents a temporary storage carbohydrate in early-spring deadnettle, at the same time perhaps functioning as a membrane protector and/or as an antioxidant in the vicinity of membranes, as recently suggested for other RFOs and fructans. This novel finding urges further research on this peculiar carbohydrate on a broader array of RFO accumulators.

Fig. 1.

Structures of RFOs in L. purpureum. (A) Raffinose (Raf) is an α(1 → 6) elongation of sucrose (Suc). Raf might be subjected to β-fructosidase activity leading to the production of fructose and melibiose (Mel). Alternatively, the action of α-galactosidase leads to the formation of galactose (Gal) and Suc B. Stachyose (Sta) is an α(1 → 6) elongation of Raf. Sta might be subjected to β-fructosidase activity leading to the production of fructose and manninotriose (Min). Alternatively, the action of α-galactosidase leads to the formation of galactose (Gal) and Raf.

Fig. 4.

Lamium purpureum pictures representing the dissections of (A) the eight different parts of the stem (stem 0 representing the internode between the bottom leaf and the leaf above) as well as top and bottom leaves, and (B) the four different parts of the major vein dissected from the bottom leaf.

Fig. 2.

HPAEC-PAD chromatograms of stem parts derived from Lamium purpureum, Leonurus cardiaca, Physostegia virginiana, Cornus mas, Melissa officinalis, Ajuga reptans and Lamiastrum galeobdolon as compared with a reference mixture containing 150 µm of galactinol (Gol), 150 µm of glucose (Glc), 150 µm of fructose (Fru), 150 µm of melibiose (Mel), 150 µm of manninotriose (Min), 150 µm sucrose (Suc), 150 µm of raffinose (Raf) and 150 µm of stachyose (Sta). Galactose (Gal) was not included in the reference.

Fig. 3.

HPAEC-PAD chromatograms of different parts of L. purpureum, compared with a reference mixture containing 10 µm of mannitol (Mtl), 10 µm of glucose (Glc), 10 µm of fructose (Fru), 10 µm of melibiose (Mel), 10 µm of manninotriose (Min), 10 µm of sucrose (Suc), 10 µm of raffinose (Raf) and 10 µm of stachyose (Sta). Galactose (Gal) was not included in the reference.

Fig. 5.

Carbohydrate gradients in different parts of L. purpureum stems and in top leaves (TL) and bottom leaves (BL) as illustrated in Fig. 4A. Relative values are presented and the maximal concentration of each sugar is indicated in μg f. wt⁻¹. Min, Manninotrios; Sta, stachyose; Mel, melibiose; Raf, raffinose; Glc/Gal, glucose/galactose; Suc, sucrose; Fru, fructose; Gol, galactinol. Standard errors are presented for n = 5.

Fig. 6.

HPAEC-PAD chromatograms of the different vein parts 1–4 (as defined in Fig. 4B) and inter-vein area as compared with a reference mixture containing 10 µm of galactinol (Gol), 10 µm of mannitol (Mtl), 10 µm of glucose (Glc), 10 µm of fructose (Fru), 10 µm of melibiose (Mel), 10 µm of manninotriose (Min), 10 µm of sucrose (Suc), 10 µm of raffinose (Raf) and 10 µm of stachyose (Sta). Galactose (Gal) was not included in the reference.

Fig. 7.

HPAEC-PAD chromatograms of leaves treated with and without EDTA and their respective exudates, as compared with a reference mixture containing 30 µm of galactinol (Gol), 30 µm of mannitol (Mtl), 30 µm of glucose (Glc), 30 µm of fructose (Fru), 30 µm of melibiose (Mel), 30 µm of manninotriose (Min), 30 µm of sucrose (Suc), 30 µm of raffinose (Raf) and 30 µm of stachyose (Sta). Galactose (Gal) was not included in the reference. Tris, Tris(hydroxymethyl)aminomethane.