Structure of acid-extractable polysaccharides of tree greenery of Picea abies

Highlights

• The acid-extractable pectin (PA_A fraction) were isolated from P. abies greenery.

• RG-I side chains are formed out of branched 1,5-α-L-arabinan and 1,4-β-D-galactan.

• PA_A fraction contained glycans of the glucuronoarabinoxylan and glucomannan classes.

• However, neither glucomannans nor glucuronoarabinoxylans were associated with RG-I.

Abstract

The polysaccharide PA_A was isolated from tree greenery of spruce Picea abies by the extraction with HCl solution. Monosaccharide analysis PA_A revealed high content of uronic acid residues (45%), arabinose (19.5%) and galactose (8.6%). It was shown that this polysaccharide contained polymers of various structures. The major component of which were pectic polysaccharides represented mainly by partly methyl-esterified and partly acetylated 1,4-α-D-galacturonan and by minor parts of partly 2-O- and/or 3-O- acetylated rhamnogalacturonan-I (RG-I). The side carbohydrate chains of the branched region of RG-I were represented predominantly by highly branched 1,5-α-L-arabinan and minor portions of 1,4-β-D-galactan.

In addition to the dominant pectins, the isolated polysaccharide also contained arabinogalactan proteins (AGP) and binding glycans of the glucuronoarabinoxylan (GAX), glucomannan (GM) and xyloglucan classes, which indicated a close interaction of these polysaccharides in the cell walls. It can be concluded that, at least a part of the pectin is strongly associated with the AGP and binding glycans of the GAX and GM classes. However, neither GM nor GAX were associated with RG-I.

Introduction

The plant cell wall is a finely organized complex of various polysaccharides, proteins and other polymers assembled in relatively structurally independent but interacting networks. Linear microfibrils of cellulose interconnected by binding glycans are a part of the main network that forms the framework of the cell wall. The second network is represented by pectic polysaccharides. The third network is constructed of structural proteins (Alekhina et al., 2005).

The major components of pectic substances are galacturonan (HG), rhamnogalacturonan-I (RG-I), rhamnogalacturonan-II, arabinan, galactan and arabinogalactan, which indicate the high complexity of these polysaccharides (Caffall & Mohnen, 2009). HG is considered to be the most widely distributed component. Usually, it reaches more than 50% of the total pectin content (depending on the source), whereas RG-I accounts for 5–45% (Voragen, Coenen, Verhoef, & Schols, 2009; Yapo, 2011; Yapo & Gnakri, 2015).

The bulk of the arabinogalactan proteins (AGP) (up to 95%) is represented by the carbohydrate part of the molecules, consisting of type II arabinogalactan (AG-II) and 2–10% is represented by a polypeptide core containing mainly amino acids such as Hyp, Ala, Ser and Thr (Ellis, Egelund, Schultz, & Bacic, 2010).

Pectic polysaccharides and AGP are often described as interconnected cell wall polymers in various tissues of plants, including grape (Vitis vinifera) (Pellerin, Vidal, Williams, & Brillouet, 1995), spent hops Humulus lupulus (Oosterveld, Voragen, & Schols, 2002), roots of Vernonia kotschyana (Nergard et al., 2005), Beta vulgaris (McKenna, Al-Assaf, Phillips, & Funami, 2006), taproots of carrot (Daucus carota) (Immerzeel, Eppink, de Vries, Schols, & Voragen, 2006), Coffea arabica beans (Redgwell, Curti, Fischer, Nicolas, & Fay, 2002), aboveground parts of Heracleum sosnowskyi (Makarova, Shakhmatov, & Belyy, 2016), and from cell culture medium of Arabidópsis thaliána (Tan et al., 2013).

Other important components of the matrix of the plant cell wall are so-called binding glycans, such as xyloglucans, heteroxylans and heteromannans. Coniferous wood is characterized by the presence of significant amounts of mannans (up to 25%) and usually contains 5–10% of arabinoglucuronoxylan (Hilpmann et al., 2016; Willför, Sundberg, Tenkanen, & Holmbom, 2008).

Despite the fact that structure of various cell wall components is well described in the literature, some features of their structure are still being discussed. Moreover, there is no reliable knowledge on interaction of these components with each other, because the way by which the components of the cell wall are assembled into a single structural-functional ensemble is unknown.

Our previous studies established that tree greenery of Siberian fir A. sibirica, large-scale waste of the wood-processing industry, is a potential source of the pectin (with the content up to 8%), which we proposed to call “abienan” (Shakhmatov, Udoratina, Atukmaev, & Makarova, 2015).

Norway spruce (Picea abies) is an evergreen conifer tree, a species of the genus Picea in the family Pinaceae. It is known that extracts obtained from spruce have a wide range of physiological activity (Fyhrquist, Virjamo, Hiltunen, & Julkunen-Tiitto, 2017; Le Normand et al., 2014). One of the most promising areas for the use of P. abies tree greenery is the non-waste processing with the production of biologically active substances for therapeutic and prophylactic purposes.

Up to the present time, there is a lack of studies on structure of polysaccharides of tree greenery of P. abies, despite the relevance and economic importance of this raw material (Makarova, Shakhmatov, Udoratina, & Kutchin, 2015). The study of the chemical composition and structure of polysaccharides of extracts of tree greenery of P. abies could be used as a basis for development and improvement of new technologies for the comprehensive processing of this raw material and for determination of prospects for their use.

In the previous paper, the fractions containing AGP and pectic polysaccharides, as well as binding glycans of the glucuronoxylan and glucomannan classes were isolated from tree greenery of P. abies by successive extraction with water and solutions of HCl, (NH₄)₂C₂O₄, KOH and NaOH (Makarova, Shakhmatov, & Belyy, 2017). It was found that the fraction extracted with water from P. abies tree greenery consisted mainly of AGP and minor amounts of pectic polysaccharides (Makarova, Shakhmatov, & Belyy, 2018).

Most published studies on structural organization of pectin-, arabinan- and galactan-containing polysaccharides of spruce were devoted to analysis of their qualitative and quantitative monosaccharide composition. In particular, the structure of arabinogalactans and pectins from various species of spruce was studied mainly by means of methylation methods and one-dimensional NMR spectroscopy (Makarova et al., 2015). However, the structure of such complex organized polysaccharides can be studied more deeply with the use of two-dimensional NMR spectroscopy. Two-dimensional NMR makes it possible to obtain more information about molecular structure. This is particularly convenient for the elucidation of structure of complex molecules, which can be a difficult task for the one-dimensional NMR spectroscopy.

The present work was dedicated to revealing general patterns and features of structure of polysaccharides extracted with an aqueous solution of HCl from tree greenery of P. abies using the methods of structural chemistry of carbohydrates, mainly by NMR spectroscopy, which is one of the most promising physical-chemical methods of analysis. The isolated polysaccharides were studied in detail by the methods of ion exchange chromatography, partial acidic and enzymatic hydrolysis, as well as ¹H and ¹³C NMR spectroscopy using the correlation spectroscopy methods TOCSY, COSY, HSQC, HSQC-NOESY, HSQC-TOCSY, spectroscopy of the nuclear Overhauser effect in the rotating coordinate system ROESY and multiple bond correlation spectroscopy HMBC.