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Seasonal dynamics of polysaccharides in Norway spruce (Picea abies)
Carbohydrate Polymers 157 (2017) 686–694
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Seasonal dynamics of polysaccharides in Norway spruce (Picea abies) Elena N. Makarova ∗ , Evgeny G. Shakhmatov, Vladimir A. Belyy Institute of Chemistry, Komi Science Centre, Urals Branch of the Russian Academy of Sciences, Pervomaiskaya St. 48, Syktyvkar 167982, Russia
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Article history: Received 7 July 2016 Received in revised form 12 October 2016 Accepted 12 October 2016 Available online 17 October 2016 Keywords: Picea abies Abies sibirica Pectic polysaccharides Arabinogalactan Arabinogalactan protein Coniferous tree greenery
a b s t r a c t Annual dynamics of accumulation and changes in the monosaccharide composition of pectin-, arabinanand galactan-containing polysaccharides and binding glycans isolated from greenery (thin branches with needles) of Norway spruce were investigated in this study. The polysaccharides were compared with polysaccharides of Siberian fir according to the yields, composition and content of typical components. It was shown that Norway spruce greenery contains lowly methyl-esterified pectin extracted with ammonium oxalate, which is a part of protopectic complex and is bound with components of cell walls via ionic bonds. In contrast, Siberian fir greenery contains mainly water-extracted highly methyl-esterified pectin, weakly bound to cell wall components. It was concluded that an autumn-winter period is the optimal time for harvesting Norway spruce and Siberian fir greenery for isolation of the pectic polysaccharides. The revealed regularities indicate that there is a certain biorhythm of accumulation of the compounds, probably determined by genetic factors. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Forest resources in Russia, mostly formed by conifer forests, are the principal factor shaping the Russian nature and providing various useful products. The analysis of the literature on the structural chemistry of polysaccharides has shown that the polysaccharides of conifers (except cellulose and binding glycans) are the least studied polysaccharides, in spite of the demand and economic importance of this raw material (Makarova, Shakhmatov, Udoratina, & Kutchin, 2015). Phloem and cambium, sapwood and heartwood, tissues of a tree trunk, are the principal objects of studies of the polysaccharide composition of wood of conifers. The phloem and cambium are characterized by a high content of pectin (4.4–18%), while in the sapwood and heartwood its content is low (0.5–3.8%) (Makarova et al., 2015; Thornber & Northcote, 1961a, 1961b; Willför, Sundberg, Hemming, & Holmbom, 2005). The monosaccharide composition and content of water-soluble polysaccharides in the sapwood and heartwood have been studied for the most widespread types of coniferous trees belonging to the genus Abies of the Pine family (Abies balsamea, Abies sibirica, Abies lasiocarpa), larches (Larix lariciana, L. sibirica, Larix decidua, L. occidentalis), pines (Pinus banksiana, Pinus resinosa, Pinus silvestris, Pinus taeda, Pseudotsuga menziesii), spruces (Picea abies, Picea glauca,
∗ Corresponding author. E-mail address: [email protected] (E.N. Makarova). http://dx.doi.org/10.1016/j.carbpol.2016.10.035 0144-8617/© 2016 Elsevier Ltd. All rights reserved.
P. rubens, Picea mariana), and the genus Thuja in the cypress family (Thuja occidentalis). The greatest amount of pectic polysaccharides (1.5%) was isolated from wood of A. balsamea (Bertaud & Holmbom, 2004; Goellner et al., 2011; Karácsonyi, Kováˇcik, Alföldi, & Kubaˇcková, 1984; Odonmazig, Ebringerová, Machová, & Alföldi, 1994; Ponder, 1998; Willfor & Holmbom, 2004; Willför, Sjöholm, Laine, & Holmbom, 2002; Willfor et al., 2005). Complex utilization of forest resources involves the use of whole biomass of trees as well as utilization of wood waste generated during harvesting and wood processing at logging enterprises. Pectic polysaccharides were found in bark of various species of coniferous trees, e.g., Abies amabilis (Bhattacharjee & Timell, 1965), Pinus pinaster (Fradinho et al., 2002), P. sylvestris (Valentín et al., 2010), L. sibirica, Larix gmelinii (Trofimova, Medvedeva, Ivanova, Babkin, & Malkov, 2012), and P. abies (Bianchi et al., 2015; Kemppainen, Siika-Aho, Pattathil, Giovando, & Kruus, 2014; Krogell, Holmbom, Pranovich, Hemming, & Willför, 2012; Le Normand et al., 2014). Meanwhile, it is known that biological processes actively take place in storage organs of coniferous trees, particularly in needles, where metabolites, including pectins, are accumulated and spent during perennial cycles for growing of the vegetative mass (Robakidze & Bobkova, 2003). In the total amount of waste generated by different branches of wood-processing industry, the proportion of wood greenery is 20–30 million tons per year (Yagodyn, 2001). Coniferous greenery is a perspective raw material resource for production of biologically active substances for therapeutic and prophylactic applications due to the possibility of year-round using and sufficient availability of the raw material base.
E.N. Makarova et al. / Carbohydrate Polymers 157 (2017) 686–694
It is known that wood greenery contains much more pectic compounds than wood of a trunk (Shakhmatov, Udoratina, Atukmaev, & Makarova, 2015; Willfor et al., 2005). However, the data on the composition and structure of the polysaccharides of storage organs of conifers is limited up to the present time, despite the potential economic importance of this raw material (Makarova et al., 2015; Shakhmatov et al., 2015). As a result of our previous studies, it was found that greenery of Siberian fir A. sibirica is a potential source of pectin (content of up to 8%). An effective method of obtaining pectin polysaccharides and binding glycans from coniferous greenery was developed and patented on the example of A. sibirica (Patova, Makarova, & Shakhmatov, 2011). The previous studies encompass the annual dynamics of accumulation and the character of change of the monosaccharide composition of polysaccharides of greenery of A. sibirica. It was shown that an autumn period is optimal for harvesting coniferous greenery to isolate pectic polysaccharides (taken into account the content of uronic acids, protein and starch), and a winter-spring period is optimal to isolate the binding glycans (Makarova, Patova, Mikhailova, & Demin, 2011; Shakhmatov et al., 2015). Meanwhile, it is known that the monosaccharide composition and structural organization of the polymers of plant cell walls may vary not only between different species of plants, but also between different tissues of a plant. In this regard, the study of polysaccharides of Norway spruce (P. abies), a widely distributed forest species, is of great interest. Comparison of the results with those previously obtained for fir might reveal similarities and differences of these sources of biologically active substances (e.g. polysaccharides). The Norway spruce (P. abies) is a large evergreen coniferous tree of the genus Picea in the family Pinaceae. Wood greenery of P. abies, large-tonnage waste of wood-processing industry, is relatively new, insufficiently studied, non-traditional raw material to obtain pectic polysaccharides. Efficient use of spruce greenery is impeded by the lack of knowledge about the dynamics of accumulation, composition and structure of the cell wall polysaccharides. Meanwhile, a purposeful study of structural features of components of P. abies greenery and their chemical characteristics can help to develop the scientific basis of the greenery processing, which is crucial for determining prospects of application of the greenery. This study presents the analysis of the dynamics of accumulation and the character of change of monosaccharide composition of polysaccharides of P. abies greenery during a year. The yields, composition and content of typical components (uronic acid, neutral sugars) of the spruce greenery polysaccharides were compared with respective characteristics of previously studied polysaccharides of greenery of Siberian fir A. sibirica.
2. Materials and methods 2.1. Preparation of plant raw material and isolation of polysaccharides Coniferous greenery of Picea abies was collected near Syktyvkar (Komi Republic, Russia) in the period from January to December 2013 (except August). The samples were taken from 10 to 20 growing trees in the middle of each month. Such biological replication gives a representative sample providing the 5% level of significance (Sudachkova & Semenova, 1971). For analysis of coniferous greenery, thin branches (less than 8 mm in diameter) with needles, from the top, middle and bottom sections of a tree crown were cut in four different geodesic directions. The isolation was performed according to the procedure described earlier (Shakhmatov et al., 2015). The residual raw mate-
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Greenery of spruce (Picea abies) Extraction with ethyl acetate and chloroform
Low molecular weight substances
Raw material residue H2O, 70°C
Combined extract
1. Concentrating. 2. Precipitation. 3. Dialysis. 4. Freeze-drying
PAW
Combined extract
1. Concentrating. 2. Precipitation. 3. Dialysis. 4. Freeze-drying
PAA
Combined extract
1. Concentrating. 2. Precipitation. 3. Dialysis. 4. Freeze-drying
PAO
Raw material residue HCl, pH~4.0, 70°C)
Raw material residue 0.7% (COONH4)2, 70°C
Raw material residue Combined extract
1. Neutralization. 2. Concentrating. 3. Dialysis. 4. Freeze-drying
PAK
Combined extract
1. Neutralization. 2. Concentrating. 3. Dialysis. 4. Freeze-drying
PAN
7% KOH, 25°C
Raw material residue 14% NaOH, 25°C
Raw material residue
Fig. 1. Scheme of extraction of greenery of Norway spruce.
rial (50 g) was extracted five times with distilled water (1 l) under continuous stirring for 2 h at 70 ◦ C (Fig. 1). The combined extract was filtered, concentrated and centrifuged. The supernatant was collected and precipitated with four volumes of 96% ethanol. The precipitate was separated by centrifugation and redissolved in water. The resulting polysaccharide PAW was dialyzed and freezedried. Hydroalcoholic supernatants obtained by ethanol precipitation of PAW fractions were combined, concentrated and dialyzed against distilled water (3.5 kDa membrane). The resulting solution was centrifuged, concentrated and freeze-dried. As a result, polysaccharide fractions PAW -S were obtained (corresponding to the June–December period). The residual material was treated with dilute hydrochloric acid at pH ∼3.5–4 (1 l) with continuous stirring and heating to 70 ◦ C for 2 h five times. The combined extract was treated as described above. As a result, the polysaccharide PAA was obtained. Hydroalcoholic supernatants obtained by ethanol precipitation of PAA fractions were combined, concentrated and dialyzed against distilled water (3.5 kDa membrane). The resulting solution was centrifuged, concentrated and freeze-dried. As a result, polysaccharide fractions PAA -S were obtained (corresponding to the June − December period). The residue was treated with 0.7% aqueous solution of (NH4 )2 C2 O4 (1 l) with continuous stirring at 70 ◦ C for 2 h five times. The combined extract was treated as described above. As a result, the polysaccharide PAO was obtained. The residual material was extracted five times with 7.0% aqueous solution of KOH (with 10 mmol/L NaBH4 ; 0.5 l) with stirring at 25 ◦ C for 2 h. The combined extract was cooled, acidified with acetic acid to pH 5.0 and centrifuged. The purified extract was concentrated, dialyzed, centrifuged and freeze-dried. After that, the polysaccharide PAK was obtained. The residue was extracted five times with 14% aqueous solution of NaOH (0.5 l) contained 10 mmol/L NaBH4 and 4% H3 BO3 with stirring at 25 ◦ C for 2 h. The combined extract was treated as described above for the polysaccharide PAK . As a result, the polysaccharide PAN was obtained. 2.2. General analytical methods The glycuronic acid content was determined by reaction with 3,5-dimethylphenol in the presence of concentrated sulphuric acid. A calibration plot was constructed for D-galacturonic acid
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12
3
11 10
2
Yields (%)
Yields (%)
2,5
1,5 1
9 8 7 6
0,5
5 4
0
I
I
II
III
IV
PАW
V
VI
VII
PАA
IX
PАO
X
XI
II
III
IV
V
VI
VII
IX
XII
Month
Fig. 2. Annual dynamics of the yield of pectic polysaccharides PAW (H2 O), PAA (HCl) and POO ((COONH4 )2 ).
(Sigma–Aldrich), and photocolorimetry was carried out at 400 and 450 nm (Usov, Bilan, & Klochkova, 1995). Protein concentration was determined using the Bradford procedure with bovine serum albumin (BSA) as a standard (Bradford, 1976). The degree of methylation was calculated as the molar ratio between methanol (determined by the method of Wood & Siddiqui, 1971) and uronic acids; photocolorimetry was carried out at 412 nm. Absorption spectra of the analyzed solutions were measured on a Shimadzu UV-1700 (PharmaSpec) spectrophotometer. Each experiment was run in triplicate. The presence of starch was determined by the iodine test (Nelson, 1944). Solutions were concentrated on a rotary evaporator (Heidolph, Germany) under reduced pressure at 40–45 ◦ C. The samples were centrifuged at 5000–10,000g for 10–20 min and then lyophilized from the frozen state using a Christ Alpha 2–4 LD lyophilizer. Solutions were dialyzed at (3.5 kDa molecular weight cut-off membrane) for 3 days, while periodically changing water every 4 h (10 l each time). 2.3. Analysis of monosaccharide composition Samples (3–5 mg) were hydrolyzed with 2 M TFA (1 ml) containing myo-inositol as an internal standard (1 mg/mL) at 100 ◦ C for 5 h. The mixture of neutral monosaccharides was converted to alditol acetates (York, Darvill, McNeil, Stevenson, & Albersheim, 1986) and identified by gas chromatography (GC) on the Shimadzu GC-2010AF chromatograph equipped with a flame ionisation detector, using an HP-1 capillary column (Agilent, 30 m × 0.25 mm × 0.25 mcm). Helium was used as the carrier gas. GC of alditol acetates was carried out at temperature programmed from 175 ◦ C (1 min) to 250 ◦ C (2 min) with a rate of 3 ◦ C/min. The content of monosaccharides as a percent of the total mass was calculated from the peak areas using coefficients of detector response. 3. Results and discussion 3.1. Dynamics of yield of polysaccharides The polysaccharides of Norway spruce and Siberian fir greenery were extracted using the similar scheme (Fig. 1). Freshly harvested Norway spruce greenery was sequentially extracted to remove extractives with ethyl acetate and chloroform. The yield of the air-dry fat-free residue was 32 − 47% (Supplemental Table 1). Thus, the pectin-containing polysaccharides (piceans) PAW, PAA , PAO (Fig. 2), and binding glycans PAK and PAN (Fig. 3) were obtained by sequential extracting of the plant raw material (Supplemental Table 2).
X
XI
XII
Month
PАK
PАN
Fig. 3. Annual dynamics of the yield of binding glycans PAK (KOH) and PAN (NaOH).
The annual dynamics of the yields of pectin-containing polysaccharides and binding glycans were studied for determination of the optimal time of harvest of spruce greenery. The structural changes related to the formation and maturation of greenery tissues are accompanied with changes in the direction of biochemical processes in the plant, in particular in the dynamics of the carbohydrate content (Robakidze & Bobkova, 2003). It was shown that greenery of P. abies contains mainly pectic polysaccharides, which are extractable with ammonium oxalate (PAO ) and water (PAW ). Their yields ranged from 1 to 3%, depending on the month (Fig. 2). The pectic polysaccharide PAA , extracted by an aqueous solution of HCl, was a minor fraction with the yield of 0.5-2%. Aqueous solutions of potassium and sodium hydroxides extracted mainly binding glycans of glucuronoxylan (PAK ) and glucuronomannan (PAN ) classes, respectively. The yields of the polysaccharides PAK and PAN varied from 5 to 11% (Fig. 3). In the earlier study of the annual dynamics of yield of polysaccharides of greenery of A. sibirica, it was established that the greenery contains mainly water extracted pectic polysaccharides (the yield was up to 6.5%); the polysaccharides of the glucuronoxylan class extracted with KOH solution dominated among the binding glycans. The maximum content of pectin-containing polysaccharides in A. sibirica was observed in January, May, July and November (Makarova et al., 2011; Shakhmatov et al., 2015). Significant changes in the composition of carbohydrates of conifers occur during formation of shoots, which indicates a close relationship of carbohydrate metabolism with the growth processes. The fractions PAW, PAA , PAO from greenery of P. Abies exhibited the maximum content of pectic polysaccharides at the stage of bud development (May) and in June, the period of active growth processes (Fig. 2.). This phenomenon can be explained by the rapid accumulation of carbohydrates in greenery during the transition to summer vegetation due to intensive photosynthesis. The yield of binding glycans of the glucuronoxylan class reached 11% in these periods, while the yield of binding glycans of the glucuronomannan class was practically independent of the time of harvest (Fig. 3). The analysis of the dynamics of water-soluble polysaccharides accumulation in developing greenery of Siberian spruce P. obovata showed that the largest yield (∼1.9%) was observed in June, when the needles were actively growing in length. At the same time, the largest yield (∼2.0%) of the polysaccharides from two- and threeyear old needles was observed during the stage of bud development (May) (Robakidze & Bobkova, 2003). The consumption of the carbohydrates was observed in July (Figs. 2 and 3), when the growth of needles in length had been completed and the stage of formation of mature needles began. The similar regularities have been previously revealed for Siberian
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Fig. 4. Annual dynamics of quantitative and qualitative monosaccharide composition of water-extracted polysaccharides of greenery of P. abies (PAW ) and A. sibirica.
spruce P. obovata. It was shown that the carbohydrates were consumed more actively, when the air temperature had a positive effect on the processes of growth and development of greenery. The content of water-soluble polysaccharides in growing greenery was significantly reduced in the period of intensive accumulation of the mass of greenery (July) (Robakidze & Bobkova, 2003). The winter period is characterized by the transition of woody plants to the state of dormancy. In the period of “organic” and “deep” dormancy (September–December), changes in the carbohydrate metabolism are primarily caused by the physiological state of a tree, the influence of endogenous factors. After the transition to the state of “forced” dormancy (December–February), the influence of external factors, such as light and temperature, is intensified. A sharp decline of temperature and shortening of the length of daylight result in complete inhibition of photosynthesis (Robakidze, 2001). The yield of pectic polysaccharides and binding glycans from greenery of P. abies was significantly reduced, as the growth processes were attenuated and stopped in the autumn–winter period (September–December) (Figs. 2 and 3). During the period from January to February, pectic polysaccharides and binding glycans were gradually accumulated in the greenery. The plants need this to survive the cold period. Thus, it was shown that the accumulation of the polysaccharides in P. abies greenery is strongly associated with the growing phase and air temperature; therefore it depends on the season. The similar conclusion was made earlier in the study of the three-year dynamics of mixed-age needles of Siberian spruce. The authors have found
that the accumulation of carbohydrates depends mainly on the vegetation period of the needles and, to a lesser extent, on the year of the observations (Robakidze, 2001). 3.2. Dynamics of qualitative and quantitative monosaccharide composition of pectic polysaccharides The study of annual dynamics of qualitative and quantitative monosaccharide composition of the polysaccharides PAW revealed that the maximum content of uronic acids (degree of methylation DM 74%) in the picean PAW , on average 37%, was observed in the period of June–December, whereas the minimum content of 25% was observed in May. The maximum content of arabinose (Ara) and galactose (Gal) residues was observed in the winter–spring period (January–April) and reached ∼14%. The content of Ara and Gal residues was reduced to 5–7% in the spring-summer period (May–June). The PAW fractions are characterized by significant content of AGII (as it was proved by NMR spectroscopy), which is often present in the form of AGP. These fractions also contain highly methylesterified pectic polysaccharides (Fig. 4, Supplemental Table 3). Arabinogalactan proteins were previously found in the callus cells of spruce P. abies (Egertsdotter & Arnold 1995; Karácsonyi, ´ & Kubaˇcková, 1998), seeds and seedlings of loblolly pine Pätoprsty, P. taeda and Douglas fir (Bobalek & Johnson 1983; Loopstra, Puryear, & No, 2000), xylem of P. taeda (Yang, Wang, Sathyan, Stasolla, & Loopstra, 2005; Zhang et al., 2003) and Pinus radiata (Putoczki et al., 2007).
Fig. 5. Annual dynamics of quantitative and qualitative monosaccharide composition of polysaccharides of greenery of P. abies (PAA ) and A. sibirica extracted with aqueous hydrochloric acid.
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Fig. 6. Annual dynamics of quantitative and qualitative monosaccharide composition of polysaccharides of greenery of P. abies (PAO ) and A. sibirica extracted with aqueous ammonium oxalate.
A significant amount of mannose (Man) and glucose (Glc) in the picean PAW can indicate the presence of water-soluble glucomannan or galactolucomannan (GGM) (Fig. 4). It is known that GGM can be in both water-soluble and alkali-soluble form in wood of conifers. For example, glucomannans from Norway spruce wood extracted with hot water had the Man:Glc residues ratio of (3.5–4.1):1 (Dudkin, Gromov, Vedernikov, Katkevich, & Cherno, 1991). In O-acetylated GGM extracted with hot water from spruce thermomechanical pulp, the ratio of Man:Glc residues was 10:1.9–2.6. Approximately each 10th mannose residue was substituted at C-6 by a terminal residue of ␣-Galp (Hannuksela & Penhoat, 2004). The study of annual dynamics of monosaccharide composition of water-extracted polysaccharides of A. sibirica greenery revealed that the maximum content of uronic acid residues, in average 60–65%, was observed in the July − October period. This is significantly higher compared to water-extracted polysaccharides from P. abies (Fig. 4). The content of Ara and Gal residues is virtually independent of the time of harvest. The content of Ara residues in A. sibirica is significantly higher than that in P. abies, especially during the period of May–June. NMR spectroscopy showed that the water-extracted fraction from A. sibirica greenery represented highly methyl-esterified pectin (DM 61%) (Makarova et al., 2011; Shakhmatov et al., 2015). It is interesting to note that the content of Glc residues in A. sibirica in the summer period increased to 8%, while the content of Glc residues in P. abies reached 40%. The presence of starch in these samples was revealed by the reaction with iodine. The content of uronic acid residues (DM 75%) in the picean PAA was practically independent of the time of harvest. This content, on average 54%, with a maximum in the period from November to January (58%), indicated the presence of pectic polysaccharides (Fig. 5, Supplemental Table 4). The maximum content of arabinose residues was observed in winter and spring (December–April) and reached ∼ 17%. The minimum content (∼11%) was observed in spring and summer (May-June). The content of galactose residues was also reduced in May to 4.4%, while the content of Glc residues increased to ∼ 17% during this period. The fractions PAA are characterized by significant content of highly methyl-esterified pectic polysaccharides and AG-II (proved by NMR spectroscopy), which is often present as AGP (Fig. 5, Supplemental Table 4). In the polysaccharide extracted with aqueous HCl from greenery of A. sibirica, the maximum content of uronic acid residues was observed in the period of September − October, with an average of 55–65% (Fig. 5). The content of Ara and Gal residues was also
reduced to 3% in spring and summer (Makarova et al., 2011). It should be noted that the content of Ara residues in P. abies was substantially higher (on 6–13%) than that in A. sibirica, on the contrary, the content of Gal residues was lower, especially in the winterspring period. NMR spectroscopy showed that this fraction from A. sibirica represented lowly methyl-esterified pectin (DM 38%) (Shakhmatov et al., 2015). The content of Glc residues in A. sibirica in the spring-summer period reached 53%, which was significantly higher compared with the same fraction isolated from P. abies with the Glc content of ∼17%. Starch was also present in these samples, according to reaction with iodine. Pectic substances are known to exist in plant cell walls as independent constituents, such as arabinogalactans, galacturonan and rhamnogalacturonan, and as the macromolecules with completed biosynthesis, such as heteroglycanogalacturonans (Ridley, O’Neill, & Mohnen, 2001). It is known that the neutral components produced at various stages of metabolism can be isolated during the pectic polysaccharides extraction. The structure of these neutral components generally coincides with structural elements of side chains of branched area of pectins (Makarova, Patova, Shakhmatov, Kuznetsov, & Ovodov, 2013). Isolation and determination of structure of low molecular weight components of pectin, with simpler structure than heteroglycanogalacturonan, can supplement the information about the structure of the original pectin macromolecule. 12 low molecular weight polysaccharide fractions (9–11 kDa) were isolated from aqueous-alcoholic supernatants obtained by precipitating of aqueous extracts of the piceans PAW and PAA by 4-fold volume of 96% ethanol (study of the June–December period). Probably, the isolated polysaccharides are present in spruce greenery as the separate neutral polysaccharides formed during biosynthesis or postsynthetic modification of macromolecules of the polysaccharides PAW and PAA (Makarova et al., 2013). This conclusion is based on the possibility of their separation during the isolation of the piceans PAW and PAA . The analysis of monosaccharide composition of these fractions revealed that the structure of carbohydrate chains mainly consisted of Ara (25–49%) and GliA (10–17%) residues. This fact suggests the presence of 1,5-␣-larabinan, which often represents carbohydrate side chains of RG-I (Supplemental Table 5). Determination of their structure can supplement the information about the structure of a branched area of the piceans PAW and PAA . The hydroalcoholic supernatant obtained analogously from A. sibirica greenery was presented by a pectic polysaccharide containing large regions of highly branched 1,5-a-l-arabinan in the side chains (Makarova et al., 2013).
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Fig. 7. Annual dynamics of quantitative and qualitative monosaccharide composition of polysaccharides of greenery of P. abies (PAK ) and A. sibirica extracted with solution of potassium hydroxide.
The pectic polysaccharide PAO obtained using extraction of P. abies greenery by a solution of ammonium oxalate was the highest yield component (Fig. 2). The maximum content of uronic acid residues (DM 48%) in the picean PAO , with an average of 70%, was observed in the autumn-winter period (September-December). The minimum content (40%) was observed in the period from May to June (Fig. 6, Supplemental Table 6). The maximum content of Ara and Gal residues was observed in the winter-spring period (January–March) and reached ∼ 14 and 5.7%, respectively. The content of Ara and Gal residues decreased to 3 and 6%, respectively, in spring and summer (May–June), while the content of Glc residues increased up to 39% in this period. The fractions PAO are characterized by significant content of lowly methyl-esterified pectic polysaccharides. The analysis of the monosaccharide composition revealed that the content of uronic acids increased during the sequential extractions of P. abies greenery with water, HCl solutions, and (NH4 )2 C2 O4 from (25–38%) to (40–70%). This probably indicates decrease of the AGP content and increase of the content of pectic polysaccharides in the isolated fractions (Supplemental Table 3,4,6). Thus, it can be assumed that the water extracted fractions consist mainly of AGP and minor quantities of pectic polysaccharides. The fractions extracted with HCl solutions consist of AGP and a predominant amount of pectic polysaccharides. The fractions extracted with a solution of (NH4 )2 C2 O4 are characterized by a significant prevalence of pectic polysaccharides and a minor amount of AGP. A similar dynamics was observed in the study of chemical structure of pectic polysaccharides and arabinogalactan proteins of Heracleum sosnowskyi. It has been found that the content of arabinogalactan proteins was decreasing and the content of pectic polysaccharides was increasing during the successive extraction of H. sosnowskyi with water and solutions of HCl and (NH4 )2 C2 O4 (Makarova, Shakhmatov, & Belyy, 2016). It should be noted that the pectic polysaccharide obtained by extraction of A. sibirica greenery with ammonium oxalate was a minor component with the yield of 0.4–0.8% (Makarova et al., 2011). The maximum content of uronic acids in the polysaccharide was observed in the period of September–November, with an average of 67%. The minimum content (42%) was in the period of May–June (Fig. 6). The maximum content of Gal residues was observed in the winter-spring period (January–March) and reached ∼ 20%. The content of Gal residues in A. sibirica was significantly higher than that in P. abies, however, the content of Ara residues was lower. In spring and summer, the content of Glc residues in A. sibirica reached 28%.
According to the reaction with iodine, starch was also present in these samples. The accumulation of starch in the vegetation period is associated with high photosynthetic activity of needles and low consumption of starch by other plant organs in this period. The maximum content of starch in greenery of Siberian spruce P. obovata also appears in June, within the period of intensive growth of shoots (Robakidze, 2001). In the autumn-winter period, the reserve polysaccharides are spent to improve the frost resistance of plants (Robakidze, 2001). Starch was not detected in the piceans isolated from spruce P. abies greenery collected in autumn and winter. Thus, it was shown that P. abies greenery contains lowly methylesterified pectin extracted with ammonium oxalate, which is a part of protopectic complex and is bound with components of cell walls via ionic bonds. In contrast, Siberian fir A. sibirica greenery contains mainly water-extracted highly methyl-esterified pectin, weakly bound to cell wall components. All of the conclusions mentioned previously indicate that the investigated coniferous trees have differences in the biochemical processes including biosynthesis, changes and degradation of macromolecular components. These differences are primarily determined by biological features of the species. Silkina, Vinokourova, Vinokourov, and Latypova, 2006, found that the period of increase of needle mass went faster in P. abies trees, but in a shorter period of time, compared with A. sibirica, where it went slower and longer (44 days in Picea abies and 72 days in Abies sibirica). Thus, the results have shown that the polysaccharides of spruce P. abies and fir A. sibirica have different yields, content, and monosaccharide compositions. The observed variations in yields, quantitative and qualitative monosaccharide composition of the polysaccharides are determined, above all, by a genus of coniferous tree, extraction conditions, and a phenophase of development. However, based on the dynamics of accumulation and change of monosaccharide composition of pectin-containing polysaccharides, the optimal time to harvest spruce and fir greenery is the autumn-winter period. In particular, in the fractions of pectincontaining polysaccharides, the content of the main component of the carbohydrate chain, GliA residues, reaches the maximum value (70%) in the autumn-winter period (September–December), while the starch content is reduced. This indicates that there is a certain biorhythm of accumulation of the compounds, probably determined by genetic factors, i.e. metabolic systems of a tree support the necessary reserve of carbohydrates regardless of external environmental conditions.
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Fig. 8. Annual dynamics of quantitative and qualitative monosaccharide composition of polysaccharides of greenery of P. abies (PAN ) and A. sibirica extracted with solution of sodium hydroxide.
3.3. Dynamics of qualitative and quantitative monosaccharide composition of binding glycans The study of monosaccharide composition of the binding glycans PAK depending on the season showed that the main components of their carbohydrate chains were xylose, glucose and uronic acid residues. This result probably indicates that the binding glycans belong to the glucan and glucuronoxylan classes (Fig. 7, Supplemental Table 7). The maximum content of Xyl residues, with an average of 11%, in the polysaccharide PAK was observed in winter and spring (January–April). In May, the Xyl content was reduced to 2%, while the content of Glc increased up to 42% (Fig. 7). The maximum content of uronic acid residues was observed in the period from September to December, with an average of 12% (Supplemental Table 7). The study of the monosaccharide composition of the binding glycans of the glucuronoxylan class isolated from fir greenery showed that the content of Xyl residues, the main component, varied within a one-year period from 16 to 23%. In the period from May to July, the content of Glc residues reached 36% (Fig. 7). These samples contained starch as well, according to the reaction with iodine. Based on the dynamics of accumulation and changing of the monosaccharide composition of the binding glycans of the glucuronoxylan class, it can be concluded that the optimal time for harvesting P. abies greenery is the winter-spring period. At this time, there is a maximum content of xylose residues and reduced starch content. A similar conclusion was made previously for binding glycans of the glucuronoxylan class isolated from fir greenery (Makarova et al., 2011). The carbohydrate chain of the polysaccharide PAN mainly contains mannose and glucose residues; therefore, it is classified as alkali-soluble glucomannan (Fig. 8, Supplemental Table 8). The content of mannose residues, with an average of 17%, was practically independent of harvest time. The content of glucose residues increased to ∼ 12% to the spring-summer period. The maximum content of uronic acid residues, with an average of 10%, was observed in the period of September-December. Furthermore, the analysis of monosaccharides and polysaccharides of PAK and PAN revealed a significant content of arabinose, galactose and rhamnose residues, the main components of branched regions of RG-I, which may indicate the presence of pectic polysaccharides. The monosaccharide composition of binding glycans of the galactoglucomannan class isolated from fir greenery was similar to that of wood greenery of P. abies (Fig. 8).
The dynamics of accumulation and changes of the monosaccharide composition of the binding glycans of the glucomannan class is almost independent of harvest time except summer (starch), as the content of mannose residues is practically independent of the time of collecting the plant raw material. 3.4. Dynamics of protein content Isolation of polysaccharides of P. abies greenery was accompanied by extraction of concomitant and/or bound protein, which amount varied considerably during the year in the obtained samples (Supplemental Tables 3, 4, 6, 7, 8). The protein content in the samples of piceans and binding glycans varied within a year in the ranges of 3.5–5.8% in PAW , 1.1–1.9% in PAA , 1.1–2.4% in PAO , 8.9–14.2% in PAK , and 8.8–13.9% in PAN . It is known that the protein metabolism plays a crucial role in the adaptation of plants to severe environmental factors (Sudachkova & Girs, 1977). Thus, the large amount of proteins is accumulated in the course of adaptation of plants to low temperatures. During the vegetation period, the dynamics of protein content has two notable maximums: spring (April–May), which falls on the flowering period, and autumn (November). The decrease in protein content is observed by June, when the needles have stopped growing. This decrease takes place till the end of August. The decrease of the protein concentration in young shoots during the vegetation depends on the process of tissues differentiation (Levin & Repyakh, 1984). The protein content decreases sharply in July, when the processes of growing and differentiation have completed. Since September, proteins begin to accumulate again. The minimum protein content in the samples of piceans and binding glycans was observed in the summer period, the maximum content was observed in the autumn-winter period. The similar dynamics of protein accumulation was determined previously in greenery of P. silvestris, P. obovata, L. sibirica, and A. sibirica (Makarova et al., 2011; Sudachkova & Girs, 1977). 4. Conclusion Pectin-, arabinan- and galactan-containing polysaccharides, as well as binding glycans of the glucuronoxylan and glucomannan classes were isolated by successive extraction of P. abies greenery. The annual dynamics of accumulation and the character of change of monosaccharide composition of these polymers were studied for the first time. It was found that each of the isolated polysaccharides is characterized by an individual dynamics in the annual cycle. The content of the polysaccharides depends on the intensity
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of growth and export processes occurring in the plant depending on a vegetation period. This is probably related with specific functions of the polysaccharides and various metabolic cycles in which they participate. The dynamics of the accumulation of polysaccharides is affected by both endogenous and environmental factors. It was shown that the accumulation of polysaccharides in P. abies is largely associated with the greenery growing phase, the air temperature, i.e. it depends on the season. Certainly, the actual values of the yield and composition of the polysaccharide fractions will vary in a multi-annual cycle, but the pattern of changes of the yield and composition will remain the same with some differences caused primarily by environmental factors (e.g. temperature, water regime and others). This study shows that wood greenery of P. abies, large-tonnage waste of wood-processing industry, is a potential source of pectin. The established changes in yields and compositions of polysaccharide fractions of greenery of P. abies allow purposeful extraction of polysaccharides with desired composition for further study or practical application. The data on seasonal changes in the composition and content of polysaccharides in fir greenery can be used for assessing the quality and for determination of the time to harvest the raw material, as well as the data can be used for development of new technologies for integrated use of the raw material. General regularities and features of the structure of carbohydrate chains of the polysaccharides of P. abies will be studied in future work. Acknowledgements This work was partially supported from the UB RAS Project No. 15-21-3-11. The research done using the equipment of Center for Collective Use “Khimiya” of Russian Academy of Sciences Ural Branch Komi Science Centre Institute of Chemistry. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbpol.2016.10. 035. References Bertaud, F., & Holmbom, B. (2004). Chemical composition of earlywood and latewood in Norway spruce heartwood, sapwood and transition zone wood. Wood Science and Technology, 38(4), 245–256. Bhattacharjee, S. S., & Timell, T. E. (1965). A study of the pectin present in the bark of amabilis fir (Abies amabilis). Canadian Journal of Chemistry, 43(4), 758–765. Bianchi, S., Kroslakova, I., Janzon, R., Mayer, I., Saake, B., & Pichelin, F. (2015). Characterization of condensed tannins and carbohydrates in hot water bark extracts of European softwood species. Phytochemistry, 120, 53–61. Bobalek, J. F., & Johnson, M. A. (1983). Arabinogalactan-proteins from Douglas fir and loblolly pine. Phytochemistry, 22(6), 1500–1503. Bradford, H. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principal of protein–dye-binding. Analytical Biochemistry, 72, 248–254. Dudkin, M. S., Gromov, V. S., Vedernikov, N. A., Katkevich, R. G., & Cherno, N. K. (1991). Hemicelluloses. Riga: Zinatne (in Russian) Egertsdotter, U., & Arnold, S. (1995). Importance of arabinogalactan proteins for the development of somatic embryos of Norway spruce (Picea abies). Physiologia Plantarum, 93(2), 334–345. Fradinho, D. M., Neto, C. P., Evtuguin, D., Jorge, F. C., Irle, M. A., Gil, M. H., et al. (2002). Chemical characterisation of bark and of alkaline bark extracts from maritime pine grown in Portugal. Industrial Crops and Products, 16(1), 23–32. Goellner, E. M., Utermoehlen, J., Kramer, R., & Classen, B. (2011). Structure of arabinogalactan from Larix laricina and its reactivity with antibodies directed against type-II-arabinogalactans. Carbohydrate Polymers, 86(4), 1739–1744. Hannuksela, T., & Penhoat, C. H. (2004). NMR structural determination of dissolved O-acetylated galactoglucomannan isolated from spruce thermomechanical pulp. Carbohydrate Research, 339, 301–312. Karácsonyi, Kováˇcik, V., Alföldi, J., & Kubaˇcková, M. (1984). Chemical and 13C-nmr studies of an arabinogalactan from Larix sibirica L. Carbohydrate Research, 134(2), 265–274.
693
´ V., & Kubaˇcková, M. (1998). Structural study on Karácsonyi, Pätoprsty, arabinogalactan-proteins from Picea abies L. Karst. Carbohydrate Research, 307(3), 271–279. Kemppainen, K., Siika-Aho, M., Pattathil, S., Giovando, S., & Kruus, K. (2014). Spruce bark as an industrial source of condensed tannins and non-cellulosic sugars. Industrial Crops and Products, 52, 158–168. Krogell, J., Holmbom, B., Pranovich, A., Hemming, J., & Willför, S. (2012). Extraction and chemical characterization of Norway spruce inner and outer bark. Nordic Pulp & Paper Research Journal, 27(1), 6–17. Le Normand, M., Mélida, H., Holmbom, B., Michaelsen, T. E., Inngjerdingen, M., Bulone, V., et al. (2014). Hot-water extracts from the inner bark of Norway spruce with immunomodulating activities. Carbohydrate Polymers, 101, 699–704. Levin, E. D., & Repyakh, S. M. (1984). Processing of wood greenery. Forest. prom-st (in Russian). Loopstra, C. A., Puryear, J. D., & No, E. G. (2000). Purification and cloning of an arabinogalactan-protein from xylem of loblolly pine. Planta, 210(4), 686–689. Makarova, E. N., Patova, O. A., Mikhailova, E. A., & Demin, V. A. (2011). Seasonal abundance and biological activity of wood greenery of Siberian fir Abies sibirica Ledeb. Himija Rastitel’nogo Syr’ja, 2, 35–42 (in Russian). http://www. chem.asu.ru/chemwood old/volume15/2011 02/1102 035.pdf Makarova, E. N., Patova, O. A., Shakhmatov, E. G., Kuznetsov, S. P., & Ovodov, Y. S. (2013). Structural studies of the pectic polysaccharide from Siberian fir (Abies sibirica Ledeb.). Carbohydrate Polymers, 92, 1817–1826. Makarova, E. N., Shakhmatov, E. G., Udoratina, E. V., & Kutchin, A. V. (2015). Structural and chemical charactertistics of pectins, arabinogalactans, and arabinogalactan proteins from conifers. Russian Chemical Bulletin, 64(6), 1302–1318. Makarova, E. N., Shakhmatov, E. G., & Belyy, V. A. (2016). Structural characteristics of oxalate-soluble polysaccharides of Sosnowsky’s hogweed (Heracleum sosnowskyi Manden). Carbohydrate Polymers, 153, 66–77. Nelson, N. (1944). A photometric adaptation of the Somogyi method for the determination of glucose. The Journal of Biological Chemistry, 153(2), 375–380. Odonmazig, P., Ebringerová, A., Machová, E., & Alföldi, J. (1994). Structural and molecular properties ofthe arabinogalactan isolated from Mongolian larchwood (Larix dahurica L.). Carbohydrate Research, 252, 317–324. Patova, O. A., Makarova, E. N., & Shakhmatov, E. G. (2011). Method for production of polysaccharides of coniferous trees greens. RU 2448119. Ponder, G. R. (1998). Arabinogalactan from Western larch: Part IV: Polymeric products of partial acid hydrolysis. Carbohydrate Polymers, 36, 1–14. Putoczki, T. L., Pettolino, F., Griffin, M. D., Möller, R., Gerrard, J. A., Bacic, A., et al. (2007). Characterization of the structure, expression and function of Pinus radiata D. Don arabinogalactan-proteins. Planta, 226(5), 1131–1142. Ridley, B. L., O’Neill, M. A., & Mohnen, D. (2001). Pectins: Structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry, 57, 929–967. Robakidze, E. A., & Bobkova, K. S. (2003). Carbohydrate accumulation in Siberian spruce needles of various ages. Russian Journal of Plant Physiology, 50(4), 509–515. Robakidze, E. A. (2001). Dynamics of carbohydrates in Siberian spruce needles, depending on environmental factors, Ph.D. Syktyvkar. Shakhmatov, E. G., Udoratina, E. V., Atukmaev, K. V., & Makarova, E. N. (2015). Extraction and structural characteristics of pectic polysaccharides from Abies sibirica L. Carbohydrate Polymers, 123, 228–236. Silkina, O. V., Vinokourova, R. I., Vinokourov, A. I., & Latypova, V. Z. (2006). Evaluation of ecological and physiological parameters of conifer trees and undergrowth plants Abies sibirica and Picea abies in the seasonal dynamics. Bulletin of TO REA, 1(27), 33–38. Sudachkova, N. E., & Girs, G. I. (1977). Metabolism of conifers and the formation of wood. Sudachkova, N. E., & Semenova, G. P. (1971). Soluble proteins of the cambial layer of Pinus sylvestris L. Lesovedenie. Thornber, J. P., & Northcote, D. H. (1961a). Changes in the chemical composition of a cambial cell during its differentiation into xylem and phloem tissue in trees. Biochemical Journal, 81(3), 449. Thornber, J. P., & Northcote, D. H. (1961b). Changes in the chemical composition of a cambial cell during its differentiation into xylem and phloem tissue in trees. 2. Carbohydrate constituents of each main component. Biochemical Journal, 81(3), 455. Trofimova, N. N., Medvedeva, E. N., Ivanova, N. V., Babkin, V. A., & Malkov, Y. A. (2012). Polysaccharides from larch biomass. INTECH Open Access Publisher. Usov, A. I., Bilan, M. I., & Klochkova, N. G. (1995). Polysaccharide of algae 48. Polysaccharide composition of several calcareous red algae: Isolation of alginate from Corallina pilutitara P. et R. (Rhodophyta, Corallinaceae). Botanica Marina, 38, 43–51. Valentín, L., Kluczek-Turpeinen, B., Willför, S., Hemming, J., Hatakka, A., Steffen, K., et al. (2010). Scots pine (Pinus sylvestris) bark composition and degradation byfungi: Potential substrate for bioremediation. Bioresource Technology, 101(7), 2203–2209. Willför, S., & Holmbom, B. (2004). Isolation and characterisation of water soluble polysaccharides from Norway spruce and Scots pine. Wood Science and Technology, 38(3), 173–179. Willför, S., Sjöholm, R., Laine, C., & Holmbom, B. (2002). Structural features of water-soluble arabinogalactans from Norway spruce and Scots pine heartwood. Wood Science and Technology, 36(2), 101–110.
694
E.N. Makarova et al. / Carbohydrate Polymers 157 (2017) 686–694
Willför, S., Sundberg, A., Hemming, J., & Holmbom, B. (2005). Polysaccharides in some industrially important softwood species. Wood Science and Technology, 39(4), 245–257. Wood, P., & Siddiqui, I. R. (1971). Determination of methanol and its application to measurement of pectin ester content and pectin methyl esterase activity. Analytical Biochemistry, 39(2), 418–428. Yagodyn, V. I. (2001). Wood greenery: Universal non-waste technology. Proceedings of scientists of chemical-Technology faculty of SPb SFTA (in Russian). Yang, S. H., Wang, H., Sathyan, P., Stasolla, C., & Loopstra, C. A. (2005). Real-time RT-PCR analysis of loblolly pine (Pinus taeda) arabinogalactan-protein and arabinogalactan-protein-like genes. Physiologia Plantarum, 124(1), 91–106.
York, W. S., Darvill, A. G., McNeil, M., Stevenson, T. T., & Albersheim, P. (1986). Isolation and characterization of plant cell walls and cell wall components. Methods in Enzymology, 118, 3–40. Zhang, Y., Brown, G., Whetten, R., Loopstra, C. A., Neale, D., Kieliszewski, M. J., et al. (2003). An arabinogalactan protein associated with secondary cell wall formation in differentiating xylem of loblolly pine. Plant Molecular Biology, 52(1), 91–102.
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Seasonal dynamics of polysaccharides in Norway spruce (Picea abies) Elena N. Makarova ∗ , Evgeny G. Shakhmatov, Vladimir A. Belyy Institute of Chemistry, Komi Science Centre, Urals Branch of the Russian Academy of Sciences, Pervomaiskaya St. 48, Syktyvkar 167982, Russia
a r t i c l e
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Article history: Received 7 July 2016 Received in revised form 12 October 2016 Accepted 12 October 2016 Available online 17 October 2016 Keywords: Picea abies Abies sibirica Pectic polysaccharides Arabinogalactan Arabinogalactan protein Coniferous tree greenery
a b s t r a c t Annual dynamics of accumulation and changes in the monosaccharide composition of pectin-, arabinanand galactan-containing polysaccharides and binding glycans isolated from greenery (thin branches with needles) of Norway spruce were investigated in this study. The polysaccharides were compared with polysaccharides of Siberian fir according to the yields, composition and content of typical components. It was shown that Norway spruce greenery contains lowly methyl-esterified pectin extracted with ammonium oxalate, which is a part of protopectic complex and is bound with components of cell walls via ionic bonds. In contrast, Siberian fir greenery contains mainly water-extracted highly methyl-esterified pectin, weakly bound to cell wall components. It was concluded that an autumn-winter period is the optimal time for harvesting Norway spruce and Siberian fir greenery for isolation of the pectic polysaccharides. The revealed regularities indicate that there is a certain biorhythm of accumulation of the compounds, probably determined by genetic factors. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Forest resources in Russia, mostly formed by conifer forests, are the principal factor shaping the Russian nature and providing various useful products. The analysis of the literature on the structural chemistry of polysaccharides has shown that the polysaccharides of conifers (except cellulose and binding glycans) are the least studied polysaccharides, in spite of the demand and economic importance of this raw material (Makarova, Shakhmatov, Udoratina, & Kutchin, 2015). Phloem and cambium, sapwood and heartwood, tissues of a tree trunk, are the principal objects of studies of the polysaccharide composition of wood of conifers. The phloem and cambium are characterized by a high content of pectin (4.4–18%), while in the sapwood and heartwood its content is low (0.5–3.8%) (Makarova et al., 2015; Thornber & Northcote, 1961a, 1961b; Willför, Sundberg, Hemming, & Holmbom, 2005). The monosaccharide composition and content of water-soluble polysaccharides in the sapwood and heartwood have been studied for the most widespread types of coniferous trees belonging to the genus Abies of the Pine family (Abies balsamea, Abies sibirica, Abies lasiocarpa), larches (Larix lariciana, L. sibirica, Larix decidua, L. occidentalis), pines (Pinus banksiana, Pinus resinosa, Pinus silvestris, Pinus taeda, Pseudotsuga menziesii), spruces (Picea abies, Picea glauca,
∗ Corresponding author. E-mail address: [email protected] (E.N. Makarova). http://dx.doi.org/10.1016/j.carbpol.2016.10.035 0144-8617/© 2016 Elsevier Ltd. All rights reserved.
P. rubens, Picea mariana), and the genus Thuja in the cypress family (Thuja occidentalis). The greatest amount of pectic polysaccharides (1.5%) was isolated from wood of A. balsamea (Bertaud & Holmbom, 2004; Goellner et al., 2011; Karácsonyi, Kováˇcik, Alföldi, & Kubaˇcková, 1984; Odonmazig, Ebringerová, Machová, & Alföldi, 1994; Ponder, 1998; Willfor & Holmbom, 2004; Willför, Sjöholm, Laine, & Holmbom, 2002; Willfor et al., 2005). Complex utilization of forest resources involves the use of whole biomass of trees as well as utilization of wood waste generated during harvesting and wood processing at logging enterprises. Pectic polysaccharides were found in bark of various species of coniferous trees, e.g., Abies amabilis (Bhattacharjee & Timell, 1965), Pinus pinaster (Fradinho et al., 2002), P. sylvestris (Valentín et al., 2010), L. sibirica, Larix gmelinii (Trofimova, Medvedeva, Ivanova, Babkin, & Malkov, 2012), and P. abies (Bianchi et al., 2015; Kemppainen, Siika-Aho, Pattathil, Giovando, & Kruus, 2014; Krogell, Holmbom, Pranovich, Hemming, & Willför, 2012; Le Normand et al., 2014). Meanwhile, it is known that biological processes actively take place in storage organs of coniferous trees, particularly in needles, where metabolites, including pectins, are accumulated and spent during perennial cycles for growing of the vegetative mass (Robakidze & Bobkova, 2003). In the total amount of waste generated by different branches of wood-processing industry, the proportion of wood greenery is 20–30 million tons per year (Yagodyn, 2001). Coniferous greenery is a perspective raw material resource for production of biologically active substances for therapeutic and prophylactic applications due to the possibility of year-round using and sufficient availability of the raw material base.
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It is known that wood greenery contains much more pectic compounds than wood of a trunk (Shakhmatov, Udoratina, Atukmaev, & Makarova, 2015; Willfor et al., 2005). However, the data on the composition and structure of the polysaccharides of storage organs of conifers is limited up to the present time, despite the potential economic importance of this raw material (Makarova et al., 2015; Shakhmatov et al., 2015). As a result of our previous studies, it was found that greenery of Siberian fir A. sibirica is a potential source of pectin (content of up to 8%). An effective method of obtaining pectin polysaccharides and binding glycans from coniferous greenery was developed and patented on the example of A. sibirica (Patova, Makarova, & Shakhmatov, 2011). The previous studies encompass the annual dynamics of accumulation and the character of change of the monosaccharide composition of polysaccharides of greenery of A. sibirica. It was shown that an autumn period is optimal for harvesting coniferous greenery to isolate pectic polysaccharides (taken into account the content of uronic acids, protein and starch), and a winter-spring period is optimal to isolate the binding glycans (Makarova, Patova, Mikhailova, & Demin, 2011; Shakhmatov et al., 2015). Meanwhile, it is known that the monosaccharide composition and structural organization of the polymers of plant cell walls may vary not only between different species of plants, but also between different tissues of a plant. In this regard, the study of polysaccharides of Norway spruce (P. abies), a widely distributed forest species, is of great interest. Comparison of the results with those previously obtained for fir might reveal similarities and differences of these sources of biologically active substances (e.g. polysaccharides). The Norway spruce (P. abies) is a large evergreen coniferous tree of the genus Picea in the family Pinaceae. Wood greenery of P. abies, large-tonnage waste of wood-processing industry, is relatively new, insufficiently studied, non-traditional raw material to obtain pectic polysaccharides. Efficient use of spruce greenery is impeded by the lack of knowledge about the dynamics of accumulation, composition and structure of the cell wall polysaccharides. Meanwhile, a purposeful study of structural features of components of P. abies greenery and their chemical characteristics can help to develop the scientific basis of the greenery processing, which is crucial for determining prospects of application of the greenery. This study presents the analysis of the dynamics of accumulation and the character of change of monosaccharide composition of polysaccharides of P. abies greenery during a year. The yields, composition and content of typical components (uronic acid, neutral sugars) of the spruce greenery polysaccharides were compared with respective characteristics of previously studied polysaccharides of greenery of Siberian fir A. sibirica.
2. Materials and methods 2.1. Preparation of plant raw material and isolation of polysaccharides Coniferous greenery of Picea abies was collected near Syktyvkar (Komi Republic, Russia) in the period from January to December 2013 (except August). The samples were taken from 10 to 20 growing trees in the middle of each month. Such biological replication gives a representative sample providing the 5% level of significance (Sudachkova & Semenova, 1971). For analysis of coniferous greenery, thin branches (less than 8 mm in diameter) with needles, from the top, middle and bottom sections of a tree crown were cut in four different geodesic directions. The isolation was performed according to the procedure described earlier (Shakhmatov et al., 2015). The residual raw mate-
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Greenery of spruce (Picea abies) Extraction with ethyl acetate and chloroform
Low molecular weight substances
Raw material residue H2O, 70°C
Combined extract
1. Concentrating. 2. Precipitation. 3. Dialysis. 4. Freeze-drying
PAW
Combined extract
1. Concentrating. 2. Precipitation. 3. Dialysis. 4. Freeze-drying
PAA
Combined extract
1. Concentrating. 2. Precipitation. 3. Dialysis. 4. Freeze-drying
PAO
Raw material residue HCl, pH~4.0, 70°C)
Raw material residue 0.7% (COONH4)2, 70°C
Raw material residue Combined extract
1. Neutralization. 2. Concentrating. 3. Dialysis. 4. Freeze-drying
PAK
Combined extract
1. Neutralization. 2. Concentrating. 3. Dialysis. 4. Freeze-drying
PAN
7% KOH, 25°C
Raw material residue 14% NaOH, 25°C
Raw material residue
Fig. 1. Scheme of extraction of greenery of Norway spruce.
rial (50 g) was extracted five times with distilled water (1 l) under continuous stirring for 2 h at 70 ◦ C (Fig. 1). The combined extract was filtered, concentrated and centrifuged. The supernatant was collected and precipitated with four volumes of 96% ethanol. The precipitate was separated by centrifugation and redissolved in water. The resulting polysaccharide PAW was dialyzed and freezedried. Hydroalcoholic supernatants obtained by ethanol precipitation of PAW fractions were combined, concentrated and dialyzed against distilled water (3.5 kDa membrane). The resulting solution was centrifuged, concentrated and freeze-dried. As a result, polysaccharide fractions PAW -S were obtained (corresponding to the June–December period). The residual material was treated with dilute hydrochloric acid at pH ∼3.5–4 (1 l) with continuous stirring and heating to 70 ◦ C for 2 h five times. The combined extract was treated as described above. As a result, the polysaccharide PAA was obtained. Hydroalcoholic supernatants obtained by ethanol precipitation of PAA fractions were combined, concentrated and dialyzed against distilled water (3.5 kDa membrane). The resulting solution was centrifuged, concentrated and freeze-dried. As a result, polysaccharide fractions PAA -S were obtained (corresponding to the June − December period). The residue was treated with 0.7% aqueous solution of (NH4 )2 C2 O4 (1 l) with continuous stirring at 70 ◦ C for 2 h five times. The combined extract was treated as described above. As a result, the polysaccharide PAO was obtained. The residual material was extracted five times with 7.0% aqueous solution of KOH (with 10 mmol/L NaBH4 ; 0.5 l) with stirring at 25 ◦ C for 2 h. The combined extract was cooled, acidified with acetic acid to pH 5.0 and centrifuged. The purified extract was concentrated, dialyzed, centrifuged and freeze-dried. After that, the polysaccharide PAK was obtained. The residue was extracted five times with 14% aqueous solution of NaOH (0.5 l) contained 10 mmol/L NaBH4 and 4% H3 BO3 with stirring at 25 ◦ C for 2 h. The combined extract was treated as described above for the polysaccharide PAK . As a result, the polysaccharide PAN was obtained. 2.2. General analytical methods The glycuronic acid content was determined by reaction with 3,5-dimethylphenol in the presence of concentrated sulphuric acid. A calibration plot was constructed for D-galacturonic acid
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12
3
11 10
2
Yields (%)
Yields (%)
2,5
1,5 1
9 8 7 6
0,5
5 4
0
I
I
II
III
IV
PАW
V
VI
VII
PАA
IX
PАO
X
XI
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III
IV
V
VI
VII
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Month
Fig. 2. Annual dynamics of the yield of pectic polysaccharides PAW (H2 O), PAA (HCl) and POO ((COONH4 )2 ).
(Sigma–Aldrich), and photocolorimetry was carried out at 400 and 450 nm (Usov, Bilan, & Klochkova, 1995). Protein concentration was determined using the Bradford procedure with bovine serum albumin (BSA) as a standard (Bradford, 1976). The degree of methylation was calculated as the molar ratio between methanol (determined by the method of Wood & Siddiqui, 1971) and uronic acids; photocolorimetry was carried out at 412 nm. Absorption spectra of the analyzed solutions were measured on a Shimadzu UV-1700 (PharmaSpec) spectrophotometer. Each experiment was run in triplicate. The presence of starch was determined by the iodine test (Nelson, 1944). Solutions were concentrated on a rotary evaporator (Heidolph, Germany) under reduced pressure at 40–45 ◦ C. The samples were centrifuged at 5000–10,000g for 10–20 min and then lyophilized from the frozen state using a Christ Alpha 2–4 LD lyophilizer. Solutions were dialyzed at (3.5 kDa molecular weight cut-off membrane) for 3 days, while periodically changing water every 4 h (10 l each time). 2.3. Analysis of monosaccharide composition Samples (3–5 mg) were hydrolyzed with 2 M TFA (1 ml) containing myo-inositol as an internal standard (1 mg/mL) at 100 ◦ C for 5 h. The mixture of neutral monosaccharides was converted to alditol acetates (York, Darvill, McNeil, Stevenson, & Albersheim, 1986) and identified by gas chromatography (GC) on the Shimadzu GC-2010AF chromatograph equipped with a flame ionisation detector, using an HP-1 capillary column (Agilent, 30 m × 0.25 mm × 0.25 mcm). Helium was used as the carrier gas. GC of alditol acetates was carried out at temperature programmed from 175 ◦ C (1 min) to 250 ◦ C (2 min) with a rate of 3 ◦ C/min. The content of monosaccharides as a percent of the total mass was calculated from the peak areas using coefficients of detector response. 3. Results and discussion 3.1. Dynamics of yield of polysaccharides The polysaccharides of Norway spruce and Siberian fir greenery were extracted using the similar scheme (Fig. 1). Freshly harvested Norway spruce greenery was sequentially extracted to remove extractives with ethyl acetate and chloroform. The yield of the air-dry fat-free residue was 32 − 47% (Supplemental Table 1). Thus, the pectin-containing polysaccharides (piceans) PAW, PAA , PAO (Fig. 2), and binding glycans PAK and PAN (Fig. 3) were obtained by sequential extracting of the plant raw material (Supplemental Table 2).
X
XI
XII
Month
PАK
PАN
Fig. 3. Annual dynamics of the yield of binding glycans PAK (KOH) and PAN (NaOH).
The annual dynamics of the yields of pectin-containing polysaccharides and binding glycans were studied for determination of the optimal time of harvest of spruce greenery. The structural changes related to the formation and maturation of greenery tissues are accompanied with changes in the direction of biochemical processes in the plant, in particular in the dynamics of the carbohydrate content (Robakidze & Bobkova, 2003). It was shown that greenery of P. abies contains mainly pectic polysaccharides, which are extractable with ammonium oxalate (PAO ) and water (PAW ). Their yields ranged from 1 to 3%, depending on the month (Fig. 2). The pectic polysaccharide PAA , extracted by an aqueous solution of HCl, was a minor fraction with the yield of 0.5-2%. Aqueous solutions of potassium and sodium hydroxides extracted mainly binding glycans of glucuronoxylan (PAK ) and glucuronomannan (PAN ) classes, respectively. The yields of the polysaccharides PAK and PAN varied from 5 to 11% (Fig. 3). In the earlier study of the annual dynamics of yield of polysaccharides of greenery of A. sibirica, it was established that the greenery contains mainly water extracted pectic polysaccharides (the yield was up to 6.5%); the polysaccharides of the glucuronoxylan class extracted with KOH solution dominated among the binding glycans. The maximum content of pectin-containing polysaccharides in A. sibirica was observed in January, May, July and November (Makarova et al., 2011; Shakhmatov et al., 2015). Significant changes in the composition of carbohydrates of conifers occur during formation of shoots, which indicates a close relationship of carbohydrate metabolism with the growth processes. The fractions PAW, PAA , PAO from greenery of P. Abies exhibited the maximum content of pectic polysaccharides at the stage of bud development (May) and in June, the period of active growth processes (Fig. 2.). This phenomenon can be explained by the rapid accumulation of carbohydrates in greenery during the transition to summer vegetation due to intensive photosynthesis. The yield of binding glycans of the glucuronoxylan class reached 11% in these periods, while the yield of binding glycans of the glucuronomannan class was practically independent of the time of harvest (Fig. 3). The analysis of the dynamics of water-soluble polysaccharides accumulation in developing greenery of Siberian spruce P. obovata showed that the largest yield (∼1.9%) was observed in June, when the needles were actively growing in length. At the same time, the largest yield (∼2.0%) of the polysaccharides from two- and threeyear old needles was observed during the stage of bud development (May) (Robakidze & Bobkova, 2003). The consumption of the carbohydrates was observed in July (Figs. 2 and 3), when the growth of needles in length had been completed and the stage of formation of mature needles began. The similar regularities have been previously revealed for Siberian
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689
Fig. 4. Annual dynamics of quantitative and qualitative monosaccharide composition of water-extracted polysaccharides of greenery of P. abies (PAW ) and A. sibirica.
spruce P. obovata. It was shown that the carbohydrates were consumed more actively, when the air temperature had a positive effect on the processes of growth and development of greenery. The content of water-soluble polysaccharides in growing greenery was significantly reduced in the period of intensive accumulation of the mass of greenery (July) (Robakidze & Bobkova, 2003). The winter period is characterized by the transition of woody plants to the state of dormancy. In the period of “organic” and “deep” dormancy (September–December), changes in the carbohydrate metabolism are primarily caused by the physiological state of a tree, the influence of endogenous factors. After the transition to the state of “forced” dormancy (December–February), the influence of external factors, such as light and temperature, is intensified. A sharp decline of temperature and shortening of the length of daylight result in complete inhibition of photosynthesis (Robakidze, 2001). The yield of pectic polysaccharides and binding glycans from greenery of P. abies was significantly reduced, as the growth processes were attenuated and stopped in the autumn–winter period (September–December) (Figs. 2 and 3). During the period from January to February, pectic polysaccharides and binding glycans were gradually accumulated in the greenery. The plants need this to survive the cold period. Thus, it was shown that the accumulation of the polysaccharides in P. abies greenery is strongly associated with the growing phase and air temperature; therefore it depends on the season. The similar conclusion was made earlier in the study of the three-year dynamics of mixed-age needles of Siberian spruce. The authors have found
that the accumulation of carbohydrates depends mainly on the vegetation period of the needles and, to a lesser extent, on the year of the observations (Robakidze, 2001). 3.2. Dynamics of qualitative and quantitative monosaccharide composition of pectic polysaccharides The study of annual dynamics of qualitative and quantitative monosaccharide composition of the polysaccharides PAW revealed that the maximum content of uronic acids (degree of methylation DM 74%) in the picean PAW , on average 37%, was observed in the period of June–December, whereas the minimum content of 25% was observed in May. The maximum content of arabinose (Ara) and galactose (Gal) residues was observed in the winter–spring period (January–April) and reached ∼14%. The content of Ara and Gal residues was reduced to 5–7% in the spring-summer period (May–June). The PAW fractions are characterized by significant content of AGII (as it was proved by NMR spectroscopy), which is often present in the form of AGP. These fractions also contain highly methylesterified pectic polysaccharides (Fig. 4, Supplemental Table 3). Arabinogalactan proteins were previously found in the callus cells of spruce P. abies (Egertsdotter & Arnold 1995; Karácsonyi, ´ & Kubaˇcková, 1998), seeds and seedlings of loblolly pine Pätoprsty, P. taeda and Douglas fir (Bobalek & Johnson 1983; Loopstra, Puryear, & No, 2000), xylem of P. taeda (Yang, Wang, Sathyan, Stasolla, & Loopstra, 2005; Zhang et al., 2003) and Pinus radiata (Putoczki et al., 2007).
Fig. 5. Annual dynamics of quantitative and qualitative monosaccharide composition of polysaccharides of greenery of P. abies (PAA ) and A. sibirica extracted with aqueous hydrochloric acid.
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Fig. 6. Annual dynamics of quantitative and qualitative monosaccharide composition of polysaccharides of greenery of P. abies (PAO ) and A. sibirica extracted with aqueous ammonium oxalate.
A significant amount of mannose (Man) and glucose (Glc) in the picean PAW can indicate the presence of water-soluble glucomannan or galactolucomannan (GGM) (Fig. 4). It is known that GGM can be in both water-soluble and alkali-soluble form in wood of conifers. For example, glucomannans from Norway spruce wood extracted with hot water had the Man:Glc residues ratio of (3.5–4.1):1 (Dudkin, Gromov, Vedernikov, Katkevich, & Cherno, 1991). In O-acetylated GGM extracted with hot water from spruce thermomechanical pulp, the ratio of Man:Glc residues was 10:1.9–2.6. Approximately each 10th mannose residue was substituted at C-6 by a terminal residue of ␣-Galp (Hannuksela & Penhoat, 2004). The study of annual dynamics of monosaccharide composition of water-extracted polysaccharides of A. sibirica greenery revealed that the maximum content of uronic acid residues, in average 60–65%, was observed in the July − October period. This is significantly higher compared to water-extracted polysaccharides from P. abies (Fig. 4). The content of Ara and Gal residues is virtually independent of the time of harvest. The content of Ara residues in A. sibirica is significantly higher than that in P. abies, especially during the period of May–June. NMR spectroscopy showed that the water-extracted fraction from A. sibirica greenery represented highly methyl-esterified pectin (DM 61%) (Makarova et al., 2011; Shakhmatov et al., 2015). It is interesting to note that the content of Glc residues in A. sibirica in the summer period increased to 8%, while the content of Glc residues in P. abies reached 40%. The presence of starch in these samples was revealed by the reaction with iodine. The content of uronic acid residues (DM 75%) in the picean PAA was practically independent of the time of harvest. This content, on average 54%, with a maximum in the period from November to January (58%), indicated the presence of pectic polysaccharides (Fig. 5, Supplemental Table 4). The maximum content of arabinose residues was observed in winter and spring (December–April) and reached ∼ 17%. The minimum content (∼11%) was observed in spring and summer (May-June). The content of galactose residues was also reduced in May to 4.4%, while the content of Glc residues increased to ∼ 17% during this period. The fractions PAA are characterized by significant content of highly methyl-esterified pectic polysaccharides and AG-II (proved by NMR spectroscopy), which is often present as AGP (Fig. 5, Supplemental Table 4). In the polysaccharide extracted with aqueous HCl from greenery of A. sibirica, the maximum content of uronic acid residues was observed in the period of September − October, with an average of 55–65% (Fig. 5). The content of Ara and Gal residues was also
reduced to 3% in spring and summer (Makarova et al., 2011). It should be noted that the content of Ara residues in P. abies was substantially higher (on 6–13%) than that in A. sibirica, on the contrary, the content of Gal residues was lower, especially in the winterspring period. NMR spectroscopy showed that this fraction from A. sibirica represented lowly methyl-esterified pectin (DM 38%) (Shakhmatov et al., 2015). The content of Glc residues in A. sibirica in the spring-summer period reached 53%, which was significantly higher compared with the same fraction isolated from P. abies with the Glc content of ∼17%. Starch was also present in these samples, according to reaction with iodine. Pectic substances are known to exist in plant cell walls as independent constituents, such as arabinogalactans, galacturonan and rhamnogalacturonan, and as the macromolecules with completed biosynthesis, such as heteroglycanogalacturonans (Ridley, O’Neill, & Mohnen, 2001). It is known that the neutral components produced at various stages of metabolism can be isolated during the pectic polysaccharides extraction. The structure of these neutral components generally coincides with structural elements of side chains of branched area of pectins (Makarova, Patova, Shakhmatov, Kuznetsov, & Ovodov, 2013). Isolation and determination of structure of low molecular weight components of pectin, with simpler structure than heteroglycanogalacturonan, can supplement the information about the structure of the original pectin macromolecule. 12 low molecular weight polysaccharide fractions (9–11 kDa) were isolated from aqueous-alcoholic supernatants obtained by precipitating of aqueous extracts of the piceans PAW and PAA by 4-fold volume of 96% ethanol (study of the June–December period). Probably, the isolated polysaccharides are present in spruce greenery as the separate neutral polysaccharides formed during biosynthesis or postsynthetic modification of macromolecules of the polysaccharides PAW and PAA (Makarova et al., 2013). This conclusion is based on the possibility of their separation during the isolation of the piceans PAW and PAA . The analysis of monosaccharide composition of these fractions revealed that the structure of carbohydrate chains mainly consisted of Ara (25–49%) and GliA (10–17%) residues. This fact suggests the presence of 1,5-␣-larabinan, which often represents carbohydrate side chains of RG-I (Supplemental Table 5). Determination of their structure can supplement the information about the structure of a branched area of the piceans PAW and PAA . The hydroalcoholic supernatant obtained analogously from A. sibirica greenery was presented by a pectic polysaccharide containing large regions of highly branched 1,5-a-l-arabinan in the side chains (Makarova et al., 2013).
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691
Fig. 7. Annual dynamics of quantitative and qualitative monosaccharide composition of polysaccharides of greenery of P. abies (PAK ) and A. sibirica extracted with solution of potassium hydroxide.
The pectic polysaccharide PAO obtained using extraction of P. abies greenery by a solution of ammonium oxalate was the highest yield component (Fig. 2). The maximum content of uronic acid residues (DM 48%) in the picean PAO , with an average of 70%, was observed in the autumn-winter period (September-December). The minimum content (40%) was observed in the period from May to June (Fig. 6, Supplemental Table 6). The maximum content of Ara and Gal residues was observed in the winter-spring period (January–March) and reached ∼ 14 and 5.7%, respectively. The content of Ara and Gal residues decreased to 3 and 6%, respectively, in spring and summer (May–June), while the content of Glc residues increased up to 39% in this period. The fractions PAO are characterized by significant content of lowly methyl-esterified pectic polysaccharides. The analysis of the monosaccharide composition revealed that the content of uronic acids increased during the sequential extractions of P. abies greenery with water, HCl solutions, and (NH4 )2 C2 O4 from (25–38%) to (40–70%). This probably indicates decrease of the AGP content and increase of the content of pectic polysaccharides in the isolated fractions (Supplemental Table 3,4,6). Thus, it can be assumed that the water extracted fractions consist mainly of AGP and minor quantities of pectic polysaccharides. The fractions extracted with HCl solutions consist of AGP and a predominant amount of pectic polysaccharides. The fractions extracted with a solution of (NH4 )2 C2 O4 are characterized by a significant prevalence of pectic polysaccharides and a minor amount of AGP. A similar dynamics was observed in the study of chemical structure of pectic polysaccharides and arabinogalactan proteins of Heracleum sosnowskyi. It has been found that the content of arabinogalactan proteins was decreasing and the content of pectic polysaccharides was increasing during the successive extraction of H. sosnowskyi with water and solutions of HCl and (NH4 )2 C2 O4 (Makarova, Shakhmatov, & Belyy, 2016). It should be noted that the pectic polysaccharide obtained by extraction of A. sibirica greenery with ammonium oxalate was a minor component with the yield of 0.4–0.8% (Makarova et al., 2011). The maximum content of uronic acids in the polysaccharide was observed in the period of September–November, with an average of 67%. The minimum content (42%) was in the period of May–June (Fig. 6). The maximum content of Gal residues was observed in the winter-spring period (January–March) and reached ∼ 20%. The content of Gal residues in A. sibirica was significantly higher than that in P. abies, however, the content of Ara residues was lower. In spring and summer, the content of Glc residues in A. sibirica reached 28%.
According to the reaction with iodine, starch was also present in these samples. The accumulation of starch in the vegetation period is associated with high photosynthetic activity of needles and low consumption of starch by other plant organs in this period. The maximum content of starch in greenery of Siberian spruce P. obovata also appears in June, within the period of intensive growth of shoots (Robakidze, 2001). In the autumn-winter period, the reserve polysaccharides are spent to improve the frost resistance of plants (Robakidze, 2001). Starch was not detected in the piceans isolated from spruce P. abies greenery collected in autumn and winter. Thus, it was shown that P. abies greenery contains lowly methylesterified pectin extracted with ammonium oxalate, which is a part of protopectic complex and is bound with components of cell walls via ionic bonds. In contrast, Siberian fir A. sibirica greenery contains mainly water-extracted highly methyl-esterified pectin, weakly bound to cell wall components. All of the conclusions mentioned previously indicate that the investigated coniferous trees have differences in the biochemical processes including biosynthesis, changes and degradation of macromolecular components. These differences are primarily determined by biological features of the species. Silkina, Vinokourova, Vinokourov, and Latypova, 2006, found that the period of increase of needle mass went faster in P. abies trees, but in a shorter period of time, compared with A. sibirica, where it went slower and longer (44 days in Picea abies and 72 days in Abies sibirica). Thus, the results have shown that the polysaccharides of spruce P. abies and fir A. sibirica have different yields, content, and monosaccharide compositions. The observed variations in yields, quantitative and qualitative monosaccharide composition of the polysaccharides are determined, above all, by a genus of coniferous tree, extraction conditions, and a phenophase of development. However, based on the dynamics of accumulation and change of monosaccharide composition of pectin-containing polysaccharides, the optimal time to harvest spruce and fir greenery is the autumn-winter period. In particular, in the fractions of pectincontaining polysaccharides, the content of the main component of the carbohydrate chain, GliA residues, reaches the maximum value (70%) in the autumn-winter period (September–December), while the starch content is reduced. This indicates that there is a certain biorhythm of accumulation of the compounds, probably determined by genetic factors, i.e. metabolic systems of a tree support the necessary reserve of carbohydrates regardless of external environmental conditions.
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Fig. 8. Annual dynamics of quantitative and qualitative monosaccharide composition of polysaccharides of greenery of P. abies (PAN ) and A. sibirica extracted with solution of sodium hydroxide.
3.3. Dynamics of qualitative and quantitative monosaccharide composition of binding glycans The study of monosaccharide composition of the binding glycans PAK depending on the season showed that the main components of their carbohydrate chains were xylose, glucose and uronic acid residues. This result probably indicates that the binding glycans belong to the glucan and glucuronoxylan classes (Fig. 7, Supplemental Table 7). The maximum content of Xyl residues, with an average of 11%, in the polysaccharide PAK was observed in winter and spring (January–April). In May, the Xyl content was reduced to 2%, while the content of Glc increased up to 42% (Fig. 7). The maximum content of uronic acid residues was observed in the period from September to December, with an average of 12% (Supplemental Table 7). The study of the monosaccharide composition of the binding glycans of the glucuronoxylan class isolated from fir greenery showed that the content of Xyl residues, the main component, varied within a one-year period from 16 to 23%. In the period from May to July, the content of Glc residues reached 36% (Fig. 7). These samples contained starch as well, according to the reaction with iodine. Based on the dynamics of accumulation and changing of the monosaccharide composition of the binding glycans of the glucuronoxylan class, it can be concluded that the optimal time for harvesting P. abies greenery is the winter-spring period. At this time, there is a maximum content of xylose residues and reduced starch content. A similar conclusion was made previously for binding glycans of the glucuronoxylan class isolated from fir greenery (Makarova et al., 2011). The carbohydrate chain of the polysaccharide PAN mainly contains mannose and glucose residues; therefore, it is classified as alkali-soluble glucomannan (Fig. 8, Supplemental Table 8). The content of mannose residues, with an average of 17%, was practically independent of harvest time. The content of glucose residues increased to ∼ 12% to the spring-summer period. The maximum content of uronic acid residues, with an average of 10%, was observed in the period of September-December. Furthermore, the analysis of monosaccharides and polysaccharides of PAK and PAN revealed a significant content of arabinose, galactose and rhamnose residues, the main components of branched regions of RG-I, which may indicate the presence of pectic polysaccharides. The monosaccharide composition of binding glycans of the galactoglucomannan class isolated from fir greenery was similar to that of wood greenery of P. abies (Fig. 8).
The dynamics of accumulation and changes of the monosaccharide composition of the binding glycans of the glucomannan class is almost independent of harvest time except summer (starch), as the content of mannose residues is practically independent of the time of collecting the plant raw material. 3.4. Dynamics of protein content Isolation of polysaccharides of P. abies greenery was accompanied by extraction of concomitant and/or bound protein, which amount varied considerably during the year in the obtained samples (Supplemental Tables 3, 4, 6, 7, 8). The protein content in the samples of piceans and binding glycans varied within a year in the ranges of 3.5–5.8% in PAW , 1.1–1.9% in PAA , 1.1–2.4% in PAO , 8.9–14.2% in PAK , and 8.8–13.9% in PAN . It is known that the protein metabolism plays a crucial role in the adaptation of plants to severe environmental factors (Sudachkova & Girs, 1977). Thus, the large amount of proteins is accumulated in the course of adaptation of plants to low temperatures. During the vegetation period, the dynamics of protein content has two notable maximums: spring (April–May), which falls on the flowering period, and autumn (November). The decrease in protein content is observed by June, when the needles have stopped growing. This decrease takes place till the end of August. The decrease of the protein concentration in young shoots during the vegetation depends on the process of tissues differentiation (Levin & Repyakh, 1984). The protein content decreases sharply in July, when the processes of growing and differentiation have completed. Since September, proteins begin to accumulate again. The minimum protein content in the samples of piceans and binding glycans was observed in the summer period, the maximum content was observed in the autumn-winter period. The similar dynamics of protein accumulation was determined previously in greenery of P. silvestris, P. obovata, L. sibirica, and A. sibirica (Makarova et al., 2011; Sudachkova & Girs, 1977). 4. Conclusion Pectin-, arabinan- and galactan-containing polysaccharides, as well as binding glycans of the glucuronoxylan and glucomannan classes were isolated by successive extraction of P. abies greenery. The annual dynamics of accumulation and the character of change of monosaccharide composition of these polymers were studied for the first time. It was found that each of the isolated polysaccharides is characterized by an individual dynamics in the annual cycle. The content of the polysaccharides depends on the intensity
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of growth and export processes occurring in the plant depending on a vegetation period. This is probably related with specific functions of the polysaccharides and various metabolic cycles in which they participate. The dynamics of the accumulation of polysaccharides is affected by both endogenous and environmental factors. It was shown that the accumulation of polysaccharides in P. abies is largely associated with the greenery growing phase, the air temperature, i.e. it depends on the season. Certainly, the actual values of the yield and composition of the polysaccharide fractions will vary in a multi-annual cycle, but the pattern of changes of the yield and composition will remain the same with some differences caused primarily by environmental factors (e.g. temperature, water regime and others). This study shows that wood greenery of P. abies, large-tonnage waste of wood-processing industry, is a potential source of pectin. The established changes in yields and compositions of polysaccharide fractions of greenery of P. abies allow purposeful extraction of polysaccharides with desired composition for further study or practical application. The data on seasonal changes in the composition and content of polysaccharides in fir greenery can be used for assessing the quality and for determination of the time to harvest the raw material, as well as the data can be used for development of new technologies for integrated use of the raw material. General regularities and features of the structure of carbohydrate chains of the polysaccharides of P. abies will be studied in future work. Acknowledgements This work was partially supported from the UB RAS Project No. 15-21-3-11. The research done using the equipment of Center for Collective Use “Khimiya” of Russian Academy of Sciences Ural Branch Komi Science Centre Institute of Chemistry. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbpol.2016.10. 035. References Bertaud, F., & Holmbom, B. (2004). Chemical composition of earlywood and latewood in Norway spruce heartwood, sapwood and transition zone wood. Wood Science and Technology, 38(4), 245–256. Bhattacharjee, S. S., & Timell, T. E. (1965). A study of the pectin present in the bark of amabilis fir (Abies amabilis). Canadian Journal of Chemistry, 43(4), 758–765. Bianchi, S., Kroslakova, I., Janzon, R., Mayer, I., Saake, B., & Pichelin, F. (2015). Characterization of condensed tannins and carbohydrates in hot water bark extracts of European softwood species. Phytochemistry, 120, 53–61. Bobalek, J. F., & Johnson, M. A. (1983). Arabinogalactan-proteins from Douglas fir and loblolly pine. Phytochemistry, 22(6), 1500–1503. Bradford, H. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principal of protein–dye-binding. Analytical Biochemistry, 72, 248–254. Dudkin, M. S., Gromov, V. S., Vedernikov, N. A., Katkevich, R. G., & Cherno, N. K. (1991). Hemicelluloses. Riga: Zinatne (in Russian) Egertsdotter, U., & Arnold, S. (1995). Importance of arabinogalactan proteins for the development of somatic embryos of Norway spruce (Picea abies). Physiologia Plantarum, 93(2), 334–345. Fradinho, D. M., Neto, C. P., Evtuguin, D., Jorge, F. C., Irle, M. A., Gil, M. H., et al. (2002). Chemical characterisation of bark and of alkaline bark extracts from maritime pine grown in Portugal. Industrial Crops and Products, 16(1), 23–32. Goellner, E. M., Utermoehlen, J., Kramer, R., & Classen, B. (2011). Structure of arabinogalactan from Larix laricina and its reactivity with antibodies directed against type-II-arabinogalactans. Carbohydrate Polymers, 86(4), 1739–1744. Hannuksela, T., & Penhoat, C. H. (2004). NMR structural determination of dissolved O-acetylated galactoglucomannan isolated from spruce thermomechanical pulp. Carbohydrate Research, 339, 301–312. Karácsonyi, Kováˇcik, V., Alföldi, J., & Kubaˇcková, M. (1984). Chemical and 13C-nmr studies of an arabinogalactan from Larix sibirica L. Carbohydrate Research, 134(2), 265–274.
693
´ V., & Kubaˇcková, M. (1998). Structural study on Karácsonyi, Pätoprsty, arabinogalactan-proteins from Picea abies L. Karst. Carbohydrate Research, 307(3), 271–279. Kemppainen, K., Siika-Aho, M., Pattathil, S., Giovando, S., & Kruus, K. (2014). Spruce bark as an industrial source of condensed tannins and non-cellulosic sugars. Industrial Crops and Products, 52, 158–168. Krogell, J., Holmbom, B., Pranovich, A., Hemming, J., & Willför, S. (2012). Extraction and chemical characterization of Norway spruce inner and outer bark. Nordic Pulp & Paper Research Journal, 27(1), 6–17. Le Normand, M., Mélida, H., Holmbom, B., Michaelsen, T. E., Inngjerdingen, M., Bulone, V., et al. (2014). Hot-water extracts from the inner bark of Norway spruce with immunomodulating activities. Carbohydrate Polymers, 101, 699–704. Levin, E. D., & Repyakh, S. M. (1984). Processing of wood greenery. Forest. prom-st (in Russian). Loopstra, C. A., Puryear, J. D., & No, E. G. (2000). Purification and cloning of an arabinogalactan-protein from xylem of loblolly pine. Planta, 210(4), 686–689. Makarova, E. N., Patova, O. A., Mikhailova, E. A., & Demin, V. A. (2011). Seasonal abundance and biological activity of wood greenery of Siberian fir Abies sibirica Ledeb. Himija Rastitel’nogo Syr’ja, 2, 35–42 (in Russian). http://www. chem.asu.ru/chemwood old/volume15/2011 02/1102 035.pdf Makarova, E. N., Patova, O. A., Shakhmatov, E. G., Kuznetsov, S. P., & Ovodov, Y. S. (2013). Structural studies of the pectic polysaccharide from Siberian fir (Abies sibirica Ledeb.). Carbohydrate Polymers, 92, 1817–1826. Makarova, E. N., Shakhmatov, E. G., Udoratina, E. V., & Kutchin, A. V. (2015). Structural and chemical charactertistics of pectins, arabinogalactans, and arabinogalactan proteins from conifers. Russian Chemical Bulletin, 64(6), 1302–1318. Makarova, E. N., Shakhmatov, E. G., & Belyy, V. A. (2016). Structural characteristics of oxalate-soluble polysaccharides of Sosnowsky’s hogweed (Heracleum sosnowskyi Manden). Carbohydrate Polymers, 153, 66–77. Nelson, N. (1944). A photometric adaptation of the Somogyi method for the determination of glucose. The Journal of Biological Chemistry, 153(2), 375–380. Odonmazig, P., Ebringerová, A., Machová, E., & Alföldi, J. (1994). Structural and molecular properties ofthe arabinogalactan isolated from Mongolian larchwood (Larix dahurica L.). Carbohydrate Research, 252, 317–324. Patova, O. A., Makarova, E. N., & Shakhmatov, E. G. (2011). Method for production of polysaccharides of coniferous trees greens. RU 2448119. Ponder, G. R. (1998). Arabinogalactan from Western larch: Part IV: Polymeric products of partial acid hydrolysis. Carbohydrate Polymers, 36, 1–14. Putoczki, T. L., Pettolino, F., Griffin, M. D., Möller, R., Gerrard, J. A., Bacic, A., et al. (2007). Characterization of the structure, expression and function of Pinus radiata D. Don arabinogalactan-proteins. Planta, 226(5), 1131–1142. Ridley, B. L., O’Neill, M. A., & Mohnen, D. (2001). Pectins: Structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry, 57, 929–967. Robakidze, E. A., & Bobkova, K. S. (2003). Carbohydrate accumulation in Siberian spruce needles of various ages. Russian Journal of Plant Physiology, 50(4), 509–515. Robakidze, E. A. (2001). Dynamics of carbohydrates in Siberian spruce needles, depending on environmental factors, Ph.D. Syktyvkar. Shakhmatov, E. G., Udoratina, E. V., Atukmaev, K. V., & Makarova, E. N. (2015). Extraction and structural characteristics of pectic polysaccharides from Abies sibirica L. Carbohydrate Polymers, 123, 228–236. Silkina, O. V., Vinokourova, R. I., Vinokourov, A. I., & Latypova, V. Z. (2006). Evaluation of ecological and physiological parameters of conifer trees and undergrowth plants Abies sibirica and Picea abies in the seasonal dynamics. Bulletin of TO REA, 1(27), 33–38. Sudachkova, N. E., & Girs, G. I. (1977). Metabolism of conifers and the formation of wood. Sudachkova, N. E., & Semenova, G. P. (1971). Soluble proteins of the cambial layer of Pinus sylvestris L. Lesovedenie. Thornber, J. P., & Northcote, D. H. (1961a). Changes in the chemical composition of a cambial cell during its differentiation into xylem and phloem tissue in trees. Biochemical Journal, 81(3), 449. Thornber, J. P., & Northcote, D. H. (1961b). Changes in the chemical composition of a cambial cell during its differentiation into xylem and phloem tissue in trees. 2. Carbohydrate constituents of each main component. Biochemical Journal, 81(3), 455. Trofimova, N. N., Medvedeva, E. N., Ivanova, N. V., Babkin, V. A., & Malkov, Y. A. (2012). Polysaccharides from larch biomass. INTECH Open Access Publisher. Usov, A. I., Bilan, M. I., & Klochkova, N. G. (1995). Polysaccharide of algae 48. Polysaccharide composition of several calcareous red algae: Isolation of alginate from Corallina pilutitara P. et R. (Rhodophyta, Corallinaceae). Botanica Marina, 38, 43–51. Valentín, L., Kluczek-Turpeinen, B., Willför, S., Hemming, J., Hatakka, A., Steffen, K., et al. (2010). Scots pine (Pinus sylvestris) bark composition and degradation byfungi: Potential substrate for bioremediation. Bioresource Technology, 101(7), 2203–2209. Willför, S., & Holmbom, B. (2004). Isolation and characterisation of water soluble polysaccharides from Norway spruce and Scots pine. Wood Science and Technology, 38(3), 173–179. Willför, S., Sjöholm, R., Laine, C., & Holmbom, B. (2002). Structural features of water-soluble arabinogalactans from Norway spruce and Scots pine heartwood. Wood Science and Technology, 36(2), 101–110.
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Willför, S., Sundberg, A., Hemming, J., & Holmbom, B. (2005). Polysaccharides in some industrially important softwood species. Wood Science and Technology, 39(4), 245–257. Wood, P., & Siddiqui, I. R. (1971). Determination of methanol and its application to measurement of pectin ester content and pectin methyl esterase activity. Analytical Biochemistry, 39(2), 418–428. Yagodyn, V. I. (2001). Wood greenery: Universal non-waste technology. Proceedings of scientists of chemical-Technology faculty of SPb SFTA (in Russian). Yang, S. H., Wang, H., Sathyan, P., Stasolla, C., & Loopstra, C. A. (2005). Real-time RT-PCR analysis of loblolly pine (Pinus taeda) arabinogalactan-protein and arabinogalactan-protein-like genes. Physiologia Plantarum, 124(1), 91–106.
York, W. S., Darvill, A. G., McNeil, M., Stevenson, T. T., & Albersheim, P. (1986). Isolation and characterization of plant cell walls and cell wall components. Methods in Enzymology, 118, 3–40. Zhang, Y., Brown, G., Whetten, R., Loopstra, C. A., Neale, D., Kieliszewski, M. J., et al. (2003). An arabinogalactan protein associated with secondary cell wall formation in differentiating xylem of loblolly pine. Plant Molecular Biology, 52(1), 91–102.