Lignans in Knotwood of Norway Spruce: Localisation with Soft X-ray Microscopy and Scanning Transmission Electron Microscopy with Energy Dispersive X-ray Spectroscopy

Tuomas Mansikkala,Andrey Pranovich,Anna Kärkönen,Sufal Swaraj,Pekka Saranpää,Riikka Piispanen,Janne Ruokolainen,Marko Huttula,Takuji Ohigashi,Risto Korpinen,Minna Patanen,Jani Seitsonen

doi:10.3390/molecules25132997

Abstract

Lignans are bioactive compounds that are especially abundant in the Norway spruce (Picea abies L. Karst.) knotwood. By combining a variety of chromatographic, spectroscopic and imaging techniques, we were able to quantify, qualify and localise the easily extractable lignans in the xylem tissue. The knotwood samples contained 15 different lignans according to the gas chromatography-mass spectrometry analysis. They comprised 16% of the knotwood dry weight and 82% of the acetone extract. The main lignans were found to be hydroxymatairesinols HMR1 and HMR2. Cryosectioned and resin-embedded ultrathin sections of the knotwood were analysed with scanning transmission X-ray microscopy (STXM). Cryosectioning was found to retain only lignan residues inside the cell lumina. In the resin-embedded samples, lignan was interpreted to be unevenly distributed inside the cell lumina, and partially confined in deposits which were either readily present in the lumina or formed when OsO4 used in staining reacted with the lignans. Furthermore, the multi-technique characterisation enabled us to obtain information on the chemical composition of the structural components of knotwood. A simple spectral analysis of the STXM data gave consistent results with the gas chromatographic methods about the relative amounts of cell wall components (lignin and polysaccharides). The STXM analysis also indicated that a torus of a bordered pit contained aromatic compounds, possibly lignin.

Highlights

In order to localise lignans in situ in Norway spruce knotwood xylem cells, concentrations of structural components and extractives in knotwood were analysed with GC mass spectrometry (GC-MS) to facilitate the analysis process with scanning transmission X-ray microscopy (STXM) and to compare quantitative results from both methods
Cellulose dominated among the other structural cell wall constituents, i.e., heteropolysaccharides and lignin (Table 1)
Norway spruce knot heartwood is an ideal research object to study how phenolic extractives and structural compounds can be distinguished in their native environment

Summary

Introduction

Norway spruce lignans are mainly composed of hydroxymatairesinol (HMR). The spruce knots contain up to 17% of HMR [4]. Lignans are optically active dimers synthesised from coniferyl alcohol, and HMR can be isolated as a mixture of two diastereomers: (7R,8R,8’R)-(−)-7-allo-hydroxymatairesinol (minor isomer) and (7S,8R,8’R)-(−)-7-hydroxymatairesinol (major isomer) [4]. Their biosynthesis is related to lignin, one of the main structural cell wall components of wood. C.K.; Cody, G.D.; Feser, M.; Jacobsen, C.; Knoll, A.H.; Wirick, S. C.K.; Zwieniecki, M.A.; Cody, G.D.; Jacobsen, C.; Wirick, S.; Knoll, A.H.; Holbrook, N.M. Evolution of xylem lignification and hydrogel transport regulation.

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