Secondary Metabolism Rearrangements in Linum usitatissimum L. after Biostimulation of Roots with COS Oligosaccharides from Fungal Cell Wall

Abstract

In vitro culture of flax (Linum usitatissimum L.) was exposed to chitosan oligosaccharides (COS) in order to investigate the effects on the growth and secondary metabolites content in roots and shoots. COS are fragments of chitosan released from the fungal cell wall during plant–pathogen interactions. They can be perceived by the plant as pathogen-associated signals, mediating local and systemic innate immune responses. In the present study, we report a novel COS oligosaccharide fraction with a degree of polymerization (DP) range of 2–10, which was produced from fungal chitosan by a thermal degradation method and purified by an alcohol-precipitation process. COS was dissolved in hydroponic medium at two different concentrations (250 and 500 mg/L) and applied to the roots of growing flax seedlings. Our observations indicated that the growth of roots and shoots decreased markedly in COS-treated flax seedlings compared to the control. In addition, the results of a metabolomics analysis showed that COS treatment induced the accumulation of (neo)lignans locally at roots, flavones luteolin C-glycosides, and chlorogenic acid in systemic responses in the shoots of flax seedlings. These phenolic compounds have been previously reported to exhibit a strong antioxidant and antimicrobial activities. COS oligosaccharides, under the conditions applied in this study (high dose treatment with a much longer exposure time), can be used to indirectly trigger metabolic response modifications in planta, especially secondary metabolism, because during fungal pathogen attack, COS oligosaccharides are among the signals exchanged between the pathogen and host plant.

Phenolic Compound Profile and Antioxidant Capacity of Flax (Linum usitatissimum L.) Harvested at Different Growth Stages

“…The most polar compound ( 1 ), with λ max of the UV spectrum at 259 nm, an [M−H] − ion at m/z 341, and an MS 2 base fragment ion of [M−H−162] − at m / z 179, was found to be coniferin. The presence of coniferin in flax leaves, and in immature seeds and roots, had previously been reported [ 9 , 15 , 20 , 32 ]. Compounds 2 and 3 showed a similar [M−H] − ion and an MS 2 base fragment ion to compound 1 , but a different UV spectrum (characteristic of caffeic acid).…”

“…Accordingly, compound 4, which showed an [M−H] − ion at m / z 609, an [M−H−120] − base fragment ion at m / z 489, an [M−H−90] − less-intense ion at m / z 519, and an [M−H−18]− ion at m / z 591, was tentatively identified as luteolin 6,8-di- C -hexoside. Since lucenin-2 (luteolin 6,8-di- C -glucoside) has already been detected in flax leaves, stems, and shoots [ 9 , 18 , 20 , 21 ], we assumed that this was compound 4 . In a similar manner, an [M−H] − ion at m / z 593, an [M−H−120] − MS 2 base ion at m / z 473, a less-intense [M−H−90] − ion at m / z 503, and an [M−H−18] − ion at m / z 575, led to the identification of compounds 6 and 8 as being apigenin 6,8-di- C -hexosides.…”

“…The structure of the former was then confirmed by comparing chromatographic and spectral data with the standard of vicenin-2 (apigenin 6,8-di- C -β- d -glucoside). Vicenin-2 had previously been found in flax leaves, stems, and shoots [ 9 , 18 , 20 , 21 ]. Compound 10 showed an [M−H] − ion at m / z 563, and [M−H−120] − , [M−H−90] − , and [M−H−60] − ions, produced from cleavages of the sugar moieties, at m / z 443, m / z 473, and m / z 503, respectively.…”

“…The parent and MS 2 ions of compound 9 were similar, although the intensity of the [M−H−60] − ion at m/z 503 was higher than that of schaftoside, and it was therefore tentatively identified as apigenin 8- C -hexoside 6- C -pentoside. Among the apigenin 6,8-di- C -glycosides with pentose and hexose moieties, schaftoside and its isomer were detected in the flax leaves and shoots [ 9 , 20 , 21 ], while vicenin-1 (apigenin-6- C -xyloside-8- C -glucoside) was identified in the leaves, stem, and shoots [ 9 , 18 , 20 , 21 ]. Moreover, three apigenin C -hexoside C -pentoside isomers were tentatively identified in the flaxseed cake [ 37 ].…”

“…Tchoumtchoua et al conducted a more thorough analysis using NMR and liquid chromatography-mass spectrometry (LC–MS) techniques in which they compared the phenolic profiles of leaves of winter and spring flax varieties, and they also detected, in addition to C -glycosyl flavones, coniferyl alcohol derivatives, although the latter was only in a small amount [ 9 ]. The effect of treating flax roots with chitosan oligosaccharides on the changes in the profile of secondary metabolites has recently been studied [ 20 ]. The authors reported the accumulation of (neo)lignans in roots, and luteolin C -glycosides and chlorogenic acid in shoots in response to stress.…”