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Partial structural characterization of pectin cell wall from Argania spinosa leaves

The pectin polysaccharides from leaves of Argania spinosa (L.) Skeels, collected from Stidia area in the west coast of northern Algeria, were investigated by using sequential extractions and the resulting fractions were analysed for monosaccharide composition and chemical structure. Water-soluble pectic (ALS-WSP) and chelating-soluble pectic (ALS-CSP) fractions were obtained, de-esterified and fractionated by anion-exchange chromatography and characterized by sugar analysis combined with methylation analysis and 1H and 13C NMR spectroscopy. The data reveal the presence of altering homogalacturonan (HG) and rhamnogalacturonan I (RG-I) in both pectin fraction. The rhamnogalacturonan I (RG-I) are consisted of a disaccharide repeating unit [→ α-D-GalpA-1,2-α-L-Rhap-1,4 →] backbone, with side chains contained highly branched α-(1 → 5)-linked arabinan and short linear β-(1 → 4)-linked galactan, attached to O-4 of the rhamnosyl residues.


Introduction
Argan tree (Argania spinosa (L.) Skeels), also known as "iron tree," is a woody species belonging to the Sapotaceae family and is endemic to Algeria and Morocco (Peltier, 1983;Baumer and Zeraia, 1999). Its geographic distribution covers a relatively large area of southwestern Algeria in northern Tindouf, where it is the second most common tree after Acacia radianna (Kaabèche et al., 2010). Some feet Argan tree is also observed in the west coast of northern Algeria (Stidia area). The argan tree is important for ecological sustainability. The woodlands protect against soil erosion and desertification owing to their deep-growing roots, they shade different types of crops, and help maintain soil fertility in arid zones (Sebaa and Kaid Harche, 2014). The argan tree also supports indigenous populations economically since almonds are used to produce argan oil (Morton and Voss, 1987), which is largely used for cooking and believed to have various medicinal properties (e.g., decreasing cholesterol level, stimulation of vascular circulation). Argan oil is also widely incorporated in many cosmetic products (Charrouf and Guillaume, 1998;Charrouf et al., 2002).
Plant cell walls are known to be potential sources of pharmacologically active polysaccharides (Gloaguen and Krausz, 2004;Paulsen, 2002). Recently, pectins, a group of anionic polysaccharides that are used in traditional pharmaceutical applications have attracted a lot of attention and have been subjected to extensive structural study (Paulsen and Barsett, 2005). Pectins are polydisperse macromolecules having high heterogeneity in terms of molecular mass and chemical structure. Their composition is affected by their origin, localization within the plant, and the extraction method used to obtain them. Pectins have a complex structure (Ridley et al., 2001), consist of three major classes namely, homogalacturonan (HG), rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II). The fine structures of the pectin occupy a biological role(s) in the cell wall. First, pectin lend strength and support to a plant and yet are very dynamic structures, especially rhamnogalacturonan type I (RG-I) which is found in the primary cell wall and provides structural support (Harholt et al., 2010). Second, pectin influences various cell wall properties such as porosity, surface charge, pH, and ion balance and therefore is of importance to the ion transport in the cell wall (Mcneil et al., 1984). Furthermore, pectin oligosaccharides are known to activate plant defense responses: they elicit the accumulation of phytoalexin which has a wide spectrum of anti-microbial activity (Hahn et al., 1981;Nothnagel et al., In this work we describe the chemical composition and structural features of pectic polysaccharides isolated from argan leaves growing in the west coast of northern Algeria (Stidia aera).

Plant material
The leaves of Argania spinosa (L.) Skeels used in this study were collected from Stidia aera (Latitude 35°49′ N; Longitude 0°02′ E), west coast of northern Algeria. After collection, leaves were dried in a ventilated oven (40°C), ground (particle size <200 μm) and stored in desiccators at room temperature.

Cell wall preparation
Fifty grams of milled powder were subjected to two successive extractions with 50:50 ethanol-toluene solutions at room temperature for 14 h. The supernatant was discarded to remove debris, cytoplasmic organelles, and starch granules.
The residue was filtered through a blotting cloth and mixed with ethanol 80% by continuous stirring for 2 h to remove any traces of toluene. Then, the residue was washed three times with distilled water and acetone solution, dried in a ventilated oven at 60°C and weighed (Harche et al., 1991).

Cell wall fractionation
Each dried residue was subjected to extraction with ethanol 80% at 90°C for 20 min. The residue was dissolved in distilled water, and the supernatant was recovered by centrifugation. The insoluble material was washed twice with distilled water at 100°C for 2 h to obtain water-soluble pectin (ALS-WSP).
The remaining residue was treated with 1% ethylenediaminetetraacetic acid (EDTA) solution at 80°C for 6 h to obtain chelating soluble pectin (ALS-CSP).
All extracts was filtered through a porous glass frit (Porosity 3) and transferred to pre-soaked dialysis tubing (Spectra/Por; molecular weight cutoff 6,000-8,000 Da). Then it was precipitated with ethanol solution (3 volumes), centrifuged and finally lyophilized.

Gas chromatography
The composition of neutral monosaccharides was determined from their alditol acetates on a Hewlett-Packard 5890A gas chromatograph (Hewlett-Packard, Palo Alto, CA, USA), equipped with a polar-fused silica capillary column (30 m × 0.53 mm) and a flame ionization detector (FID) coupled to a Article No~e00076 Hewlett-Packard 3395 integrator. Column temperature was held at 195°C for 4 min and ramped at 2.5°C·min −1 to 225°C and held for 3 min with a constant flow of 4 ml·min −1 nitrogen carrier gas. The injector and detector temperature were 260°C and 280°C, respectively. Quantification of monosaccharides was conducted using myo-inositol as an internal standard against a mixed standard solution of monosaccharides (rhamnose, fucose, xylose, arabinose, mannose, galactose, and glucose) (Selvendran et al., 1979).

Colorimetric method
The content of uronic acid was estimated using the assay of Blumenkrantz and Asboe-Hansen, (1973). The absorbance was read at 520 nm on a Beckman DU 640 spectrophotometer (Beckman Coulter, Corona, CA, USA).

Methylation analysis
Prior to methylation analysis, the carboxyl groups of the uronic acids were reduced according to the method of Taylor and Conrad, (1972). Then the polysaccharides samples were methylated twice using sodium hydroxide and methyl iodide in dry dimethyl sulfoxide, according to the method described by Hakomori, (1964). Permethylated polysaccharides were hydrolyzed with 2 N trifluoroacetic acid at 100°C for 3 h. Subsequently, they were converted into their corresponding alditol acetates by successive treatments with sodium borohydride and were acetylated with acetic anhydride in the presence of pyridine that acts as a catalyst. temperature is 260°C with a constant flow of 4 ml·min −1 nitrogen carrier gas. Column temperature was held at 165°C for 4 min and ramped at 2.5°C·min to 225°C and held for 3 min. After separation in capillary column, methylated derivatives were analyzed by a mass spectrometer (Agilent 5975C, Agilent technologies Inc., Santa Clara, CA, USA).

Nuclear magnetic resonance (NMR) spectroscopy
Samples of 15 mg were dissolved in 0.5 ml deuterated water (NMR 1 H: 5 mg in 0.5 ml solvent, NMR 13 C: 15 mg in 0.5 ml solvent, tube diameter of 5 mm) and NMR spectra were recorded on a Bruker Avance 400 spectrometer equipped with a quadruple nucleus probe at room temperature or at 60°C, at frequency 400.13 MHz for the 1 H and 100.62 MHz for 13 C. Chemical shifts δ were expressed in ppm relative to tetramethylsilane.

Yield and composition of monosaccharides
Monosaccharide extraction yield and composition of different polysaccharide fractions, determined by GC as alditol acetates, are presented in Table 1. These results revealed that the largest extract (ALS-CSP) was solubilized by EDTA (11%), the fractions solubilized by distilled water (ALS-WSP) corresponded to 5.5% of the dried material. ALS-WSP and ALS-CSP were rich in arabinose (45.3% and 75%, respectively), uronic acid (51.4% and 67.9%, respectively) and the neutral sugars were present in varying amounts.

Article No~e00076
The presence of arabinose, galactose and rhamnose suggested the lack of homogalacturonans in most of the polysaccharide fractions. Thus the high Arabinose content relative to Galactose suggests the presence of side chains of arabinan and/or arabinogalactan, while the ration of Rha/AU reported for ALS-WSP (0.20) and ALS-CSP (0.06) fractions indicating a predominance of rhamnogalacturonan-I. According to Voragen and Schols, (1996), the Rhamnogalacturonan-I can be distinguished from homogalacturonans by its ratio Rha/GalA that ranges from 0.05 to 1. These results do not exclude the possibility of the presence of rhamnogalacturonan type II as well.

Fractionation
After removal of the salts, 74.5% of ALS-WSP + and 55% of ALS-CSP + were recovered. The percentages of ALS-WSP and ALS-CSP fractions obtained with different eluting solutions are given in Table 2. These results showed that ALS-WSP and ALS-CSP fractions appear as mixtures and sub-fractions contain acidic polysaccharides. However, each of them was characterized by the

Methylation analysis of ALS-WSP(3) and ALS-CSP(3) was performed in the
carboxy-reduced sample due to the presence of galacturonic acid. The results given in Table 4 showed similar composition in both fractions, the main observed derivatives were those of arabinose, (52.7% and 60.4% respectively) which was in agreement with monosaccharide analysis. The presence of 6,6-d2-2,3,6-Me 3 -Gal-ol acetates (25.6% and 19% respectively) in approximately equal amount to the sum of the 3-Me-Rha and 3,4-Me 2 -Rha-ol acetates (15.5% and 10.6% respectively), indicated that (39.5 and 44.2% respectively) of galacturonic acid residues were involved in galacturonan blocks and (60.5 and 55.8% respectively) in rhamnogalacturonan blocks. Among the rhamnose residues, 45% and 40% respectively were substituted by arabinan or galactan side-chains (McNeil et al., 1980). The presence of high amounts of 2,3-Me2-Ara-ol acetate, indicating the presence of (1 → 5)-linked Araf units.  c Percentage of peak area relative to total peak areas, determined by GC.
3.3.3. Study by NMR spectroscopy Fig. 2 and Fig. 3 represent NMR spectra 1 H and 13 C pectic ALS-WSP(3) and ALS-CSP(3) fractions having the common characteristics of alterning homogalacturonan and rhamnogalacturonan I blocks which can be substituted by short galactan and arabinan side-chains. The chemical shift assignment was performed by comparison with spectral data already published on the structural characterization of pectins (Vriesmann and Petkowicz, 2009;Habibi et al., 2005;Rondeau-Mouro et al., 2008;Yu et al., 2010). The signals detected in the spectra correspond to the pectins and more specifically to 1,5 arabinan, 1,4 galactan, 1,4 galacturonan and 1,2 rhamnose.

Article No~e00076
The presence of arabinose and galactose in ALS-WSP(3) and ALS-CSP(3) that was detected by NMR and methylation analysis revealed two lateral arabinan and galactan branches that were separate and differed in the length of strings (arabinose stings were longer that those of galactose). In order to estimate the relative importance of the side chains to the rhamnogalacturonan backbone. The arabinose to galactose ratio is an estimation of the proportions of Ara versus Gal-rich side chains (Leivas et al., 2015). The calculated ratio was 5.0 for ALS-WSP and 8.0 for ALS-CSP demonstrating that ALS-CSP contained large arabinan side chains than ALS-WSP (16 and 10 units, respectively).
The structure of rhamnogalacturonan type I is the same in ALS-WSP(3) and ALS-CSP(3) with minor variations in arabinan side chains (Fig. 4). These   (Austarheim et al., 2014) in which rhamnogalacturonan type I structure is variable.

Conclusion
From the foregoing results it can be concluded that ALS-WSP (3)

Funding statement
This research was funded by Dr. Moulay Tahar University of Saida, Algeria.