Development of distinct cell wall layers both in primary and secondary phloem fibers of hemp (Cannabis sativa L.)

Development of distinct cell wall layers both in primary and secondary phloem fibers of hemp (Cannabis sativa L.)

Industrial Crops & Products 117 (2018) 97–109 Contents lists available at ScienceDirect Industrial Crops & Products journal homepage: www.elsevier.c...

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Industrial Crops & Products 117 (2018) 97–109

Contents lists available at ScienceDirect

Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop

Development of distinct cell wall layers both in primary and secondary phloem fibers of hemp (Cannabis sativa L.) T.E. Chernova, P.V. Mikshina, V.V. Salnikov, N.N. Ibragimova, O.V. Sautkina, T.A. Gorshkova

T ⁎

Kazan Institute of Biochemistry and Biophysics, Kazan Scientific Center, Russian Academy of Sciences, 420111, Lobachevsky Str., 2/31, Kazan, Russian Federation

A R T I C L E I N F O

A B S T R A C T

Keywords: Plant fibers G-fibers Cannabis sativa Tertiary cell wall (G-layer) Secondary cell wall Rhamnogalacturonan I

Formation of thickened cell wall allows plant fibers to obtain the strength necessary to the realization of their function as mechanical tissue. It is due to such cell wall that fibers of textile crops, like flax, hemp, and ramie, acquire characteristics that make possible to use these fibers in textile and technical applications. In the hemp stem, primary and secondary phloem fibers originating from the different type of meristems are formed. Analysis of ultrastructure coupled with immunolabelling demonstrated distinct layers within thickened cell wall in the fiber of both types: the outer layer is built as typical secondary cell wall of xylan-type, while the major portion was identified as the layers of tertiary (gelatinous or G-layer) cell wall. A newly deposited layer of the tertiary cell wall (Gn) is transformed into mature G-layer by the post-synthetic modification. The general design of cell wall in primary and secondary phloem fibers is similar, but with xylan layer being considerably thicker in secondary fibers than in primary ones. The formation of the tertiary cell wall in primary and secondary hemp fibers was associated with the synthesis of pectin component – rhamnogalacturonan I together with β-(1,4)-Dgalactan, which has been detected and characterized both in the buffer-extractable fraction and among the strongly retained within cell wall polysaccharides. Comparison of the obtained results with data on the flax fiber cell wall development permitted to find similarities, as well as some differences of G-fiber cell wall organization in different plant species.

1. Introduction Hemp is one of the oldest textile crops and has probably been grown for at least 6000 years (Small, 2015). In today's world, hemp fibers have great prospects for their use in various innovative applications as the ecological, biodegradable and renewable resource with unique properties (Ashik and Sharma, 2015; Pickering et al., 2016). Understanding of the development of the cell wall – the major component of plant fiber – is important to form the basis for further improvement of hemp fiber yield and quality. From the viewpoint of plant biology, fiber is an element of sclerenchyma, an individual cell with a thickened cell wall, reaching a length of several tens of millimeters, with a diameter of not more than a few tens of micrometers (Gorshkova et al., 2012). In the hemp stem, there are the primary phloem fibers formed from procambium and secondary phloem fibers, the result of cambium activity. Since the primary fibers are formed from the primary meristem, they appear earlier during plant biogenesis and are present from bottom to the top of the stem (Hernandez et al., 2006; Snegireva et al., 2015). Secondary

phloem fibers do not occur at the top part of the stem, they emerge closer to the stem middle, and the largest number of secondary fibers is located in the lower part of the stem. The number and occurrence of the secondary fiber along the stem length depend on the stem diameter and growth conditions of plants (Fernandez-Tendero et al., 2017). Secondary phloem fibers are known to have lower quality parameters than primary ones (Pickering et al., 2007; Placet et al., 2014). Comparison of the development of two types of phloem fibers that have the same genetic background may add understanding of the factors that determine the quality of fibers. Primary phloem fibers of hemp stem during their formation pass through the following stages of development: initiation, coordinated growth, intrusive growth and formation of the thick cell wall (Snegireva et al., 2015). The scenario for the development of secondary phloem fibers of hemp stem is the same, with the exception of the absence of coordinated elongation stage, since the formation of such fibers occurs in the stem region which has ceased to grow in length. Fibers that ceased their growth begin to thicken cell wall. Thickened cell walls of hemp fibers are often considered as



Corresponding author. E-mail addresses: [email protected] (T.E. Chernova), [email protected] (P.V. Mikshina), [email protected] (V.V. Salnikov), [email protected] (N.N. Ibragimova), [email protected] (O.V. Sautkina), [email protected] (T.A. Gorshkova). https://doi.org/10.1016/j.indcrop.2018.02.082 Received 29 November 2017; Received in revised form 29 January 2018; Accepted 28 February 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

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Fig. 1. Scheme of sample collection from hemp stem for microscopy and biochemical analysis. (A) Phloem fiber-rich strips from lower half of the stem were used for subsequent isolation of primary and secondary phloem fiber bundles for biochemistry research; samples for microscopy were collected from the 5th internode. (B) Cross-section of fiber-rich strips of hemp stem. (C) Cross-section of fiber-rich strips after detachment of secondary phloem tissue by dissecting needle. (D-G) Isolated primary and secondary phloem fiber bundles. Confocal microscopy, stained with 0.5% toluidine blue (B, C). E – epidermis, P – parenchyma cells, PF – primary fibers, SF – secondary fibers; scale bar 100 μm (B, C, E, G); 1 cm (D, F). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The important polymer of the tertiary cell wall is stage-specific rhamnogalacturonan I (RG-I) (Mikshina et al., 2013; Gorshkova et al., 2015; Gritsch et al., 2015; Guedes et al., 2017), the presence of which was not assayed in hemp fibers. The intensive post-synthetic modification of RG-I by tissue-specific galactosidase leads to transformation of cell wall structure (Roach et al., 2011) and considerable maturation of cell wall mechanical properties (Arnould et al., 2017). In flax, the nascent RG-I can be extracted from buffer-soluble fraction (Gorshkova et al., 1996). In the cell wall, the large portion of this polymer is entrapped by cellulose microfibrils and can be obtained only after their dissolution (Gurjanov et al., 2008). Both buffer-soluble polymers and those strongly retained by cellulose microfibrils were not analyzed in hemp fibers, same as the presence of post-synthetic cell wall modifications. This work aimed at revealing the ultrastructural and biochemical features of the cell wall development in primary and secondary phloem fibers in hemp stem. For this, the deposition and remodeling of G-layers of fiber cell walls in developing stems were compared for both types of fibers, the main groups of cell wall polysaccharides and β-galactosidase were immunolocalized, as well as the biochemical and structural analysis of cell wall polysaccharide fractions from isolated primary and secondary phloem fibers were carried out. The obtained results permit to designate the major part of hemp fiber cell wall as tertiary (gelatinous) cell wall and to describe its similarities and differences with other plant species.

secondary cell walls (Crônier et al., 2005; Blake et al., 2008; Dai and Fan, 2010; George et al., 2014). The characteristic features of secondary cell walls in angiosperms are the considerable proportions of xylan and lignin (Albersheim et al., 2010). Secondary cell walls often have several layers (S1-S3) that are rather similar in composition but differ in cellulose microfibril orientation. The total thickness of secondary cell walls is around 1–2 μm. Cell walls of phloem fibers in hemp seem to differ from such description. The cell wall of hemp fibers is lignified no more than 3–5.5% (McDougall et al., 1993; Garcia-Jaldon et al., 1998; Crônier et al., 2005; Fernandez-Tendero et al., 2017), with lignification extending only to the outer layers of the cell wall (Bonatti et al., 2004; Crônier et al., 2005; Fernandez-Tendero et al., 2017). The same cell wall pattern of distribution, i.e. through outer layers, was observed by immunofluorescence labeling with the antibody specific for xylan (Blake et al., 2008). The total cell wall width of hemp fibers may reach 14 μm (Crônier et al., 2005) – much higher value that is observed for secondary cell wall. Cellulose microfibrils in the main layer of hemp fiber cell wall are located almost parallel to the longitudinal axis of the cell (Dai and Fan, 2010). All those features resemble G-layer (tertiary cell wall) described for flax phloem fibers and tension wood (Mikshina et al., 2013; Gorshkova et al., 2015, Guedes et al., 2017). However, epitopes for the antibody specific for β-(1,4)-D-galactan, which labeling is characteristic for the gelatinous cell walls of fibers in flax and several other plant species (Arend, 2008; Gorshkova et al., 2015; Gritsch et al., 2015; Guedes et al., 2017), were not detected in the cell wall of hemp fibers (Blake et al., 2008), despite the high content of galactose observed in cell wall fractions biochemically extracted from phloem fiberenriched material (Crônier et al., 2005). In such samples, galactose can be the component of the parenchyma cell walls, which effectively bind antibody specific for β-(1,4)-D-galactan (Blake et al., 2008). 98

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2. Material and methods

Table 1 Antibodies used for immunocytochemistry and immunodot analysis.

2.1. Plant material The monoecious cannabinoid-free green-stem hemp (Cannabis sativa L., cultivars Diana and Antonio from the collection of the Chuvash Research Institute of Agriculture) was grown on the experimental fields of the Chuvash Research Institute of Agriculture (Tsivilsk, Russia) according to the agricultural standards. The data were similar for both Diana and Antonio varieties, the presented ones are from the analysis of Diana stems. Plants for analysis were harvested at the stage of flower formation (67-day-old plants) when stem height was 170 cm high, with top 15 cm having alternating leaf position and had 9 internodes (counting up from the cotyledons) (Fig. 1). For electron microscopy and immunocytochemistry stem segments (2 сm) were taken from the middle part of the stem (about the fifth internode). For biochemical analyses, samples were taken from the lower half of the stem, which was separated by hand into the outer part (phloem fiber-rich strips) and the inner one (xylem). After separation, phloem fiber-rich strips were immediately frozen in liquid nitrogen and then stored at −80 °C. For isolation of primary and secondary phloem fibers, the fiber-rich strips were incubated in boiling 80% ethanol for 10 min to inactivate enzymes able to degrade polysaccharides. Then, primary and secondary phloem fibers from fiber-rich strips were manually separated by dissecting needle under the stereomicroscope (Zeiss, Jena, Germany) at a magnification of × 12.5. After that, the fibers were separated from the cells of surrounding tissues (parenchyma, sieve-tube elements, and epidermis) by gentle grinding in a mortar with pestle and 80% ethanol (Fig. 1). The number of biological replicas for biochemical analyses 2, each included 3 plants.

Antibody species

Antigen/epitope

References

LM2a JIM14a INRA-RU2b LM5a LM6a

arabinogalactan protein arabinogalactan protein rhamnogalacturonan-I backbone β-(1,4)-D-galactan α-(1,5)-L-arabinan, may bind to arabinogalactan proteins

LM13a LM19a

α-(1,5)-L-arabinan (linear) homogalacturonan

LM11a LM15a LM21a N1

xylan, arabinoxylan xyloglucan mannan β-galactosidase

Yates et al. (1996) Yates et al. (1996) Ralet et al. (2010) Jones et al. 1997) Willats et al. (1998); Verhertbruggen et al. (2009a) Moller et al., (2008) Verhertbruggen et al. (2009b) McCartney et al. (2005) Marcus et al. (2008) Marcus et al. (2010) Mokshina et al. (2012)

a

Antibody was kindly provided by Prof. Paul Knox (University of Leeds, UK). Antibody was kindly provided by Dr. Fabienne Guillon (Institut National de la Recherche Agronomique, France). b

microscope operating at 80 kV and atomic resolution transmission electron microscopy complex to study nanoscale objects Hitachi HT7700 Exalens (Japan). The thickness of a cell wall layer was measured on microphotographs with known magnification. 2.3. Isolation and analysis of polysaccharide fractions Primary or secondary fibers were homogenized in liquid nitrogen with the addition of 10 mM NaOAc buffer (pH 5.0) with 0.02% NaN3. The homogenate was clarified by centrifugation at 8000g for 15 min. Clarified homogenate was incubated in boiling water bath for 10 min and mixed with 96% ethanol (the final concentration of ethanol 80%) at 4 °С overnight to precipitate the buffer-extractable polymers. The sediment was separated by centrifugation at 8000g and 4 °С for 10 min, washed three times with 80% ethanol and once with acetone and then dried. Cell wall material from pellet obtained after homogenization was isolated according to Talmadge et al. (1973). Briefly, it was sequentially washed with water (threefold), 80% ethanol, acetone (overnight, 4 °C), water (threefold) and 50 mM KH2PO4-NaOH buffer (pH 7.0) with 0.02% NaN3 (twice), digested overnight with 2 mg mL−1 glucoamylase (Sigma), washed with the same phosphate buffer, water (threefold) and acetone, and then dried. A 1-mg portion of dry cell wall was hydrolyzed by 2 M TFA at 120 °С for 1 h to determine its monosaccharide composition. Cell wall polymers were extracted sequentially by 0.5% ammonium oxalate (pH 5.0, boiling water bath, 1 h) and, after washing with water, by 4 M KOH with 3% H3BO4 (overnight). The KOH fractions were neutralized by adding acetic acid to pH 7.0. The polymers that remain after extraction with alkali, being strongly retained by cellulose microfibrils, were obtained from the residue by cellulose dissolution, according to the described method (Gurjanov et al., 2008). Briefly, cell wall residue was suspended in a solution of 8% LiCl in N,N-dimethylacetamide during 3 days. Cellulose was precipitated by water and degraded by incubation with 1% Cellusoft-L cellulase (Novo Nordisk Bioindustrie; 750 EGU/G) in 10 mM NaOAc buffer (pH 5.2) with 0.02% NaN3 at 33 °C overnight. Cell wall fractions extracted by ammonium oxalate, alkali and after cellulose digestion were desalted by passage through a Sephadex G-25 M column (8 × 270 mm), concentrated by evaporation at 60 °C, dried, hydrolyzed by 2 M TFA at 120 °C for 1 h and subjected to monosaccharide analysis. The pellet remaining after cellulose digestion was washed with water, dried, hydrolyzed by 2 M TFA, and subjected to monosaccharide analysis to evaluate “lignin-bound polymers”. The difference between the dry mass of pellet remaining after cellulose digestion and total

2.2. Transmission electron microscopy and immunocytochemistry Stem segments were infiltrated under vacuum in 0.5% glutaraldehyde in 0.05 M Na-phosphate buffer (pH 7.4) and incubated in this solution for 3 h at room temperature. Then pieces of 5 mm length and ∼1 mm width were cut out from the middle part of the pre-fixed samples and fixed in 2.5% glutaraldehyde in 0.1 M Na-phosphate buffer (pH 7.4) overnight at 4 °C. Afterwards, the samples were rinsed several times with the same buffer and post-fixed with 0.5% osmium tetroxide for 1 h. After dehydration, the samples were embedded in LR White resin (Ted Pella Inc). Ultrathin sections were cut with a diamond knife on ultramicrotome LKB Ultracut III (Sweden) and mounted on Formvarcoated 100-mesh nickel grids. Immunocytochemistry of hemp fiber was performed according to the method for wood fibers (Gorshkova et al., 2015), with minor modifications. For immunolocalization, thin sections were: (a) blocked (15 min, room temperature, high humidity chamber) in Tris-buffered saline with 0.05% Tween 20 (TBST) plus 5% normal goat serum (Sigma); (b) incubated (1.5 h at room temperature) with primary monoclonal antibodies LM11 and INRA-RU2 (Table 1) and rat IgM (Plant Probes) or polyclonal antibody N1 (Table 1) and mouse IgM (Sigma) diluted 1:200 with TBST/5% normal goat serum; (c) washed three times in 20 mM Tris-buffer (TB); (d) incubated (1.5 h at room temperature) with secondary antibody (goat anti-rat or goat anti-mouse (BBI) coupled to 5 nm colloidal gold (Amersham Pharmacia Biotech, Piscataway, NJ) diluted 1:50 in TB plus 0.06% bovine serum albumin, and (e) washed in TB and H2O. Silver enhancement for gold particles, conjugated with secondary antibody was made with the BBInternational Silver Enhancing Kit (Ted Pella Inc); the solution was applied for 2–5 min (Hainfeld and Powell, 2000). Control experiments were performed by omission of primary antibody. Sections were stained with saturated aqueous uranyl acetate followed by staining with Reynolds’ lead citrate (Reynolds, 1963). Observations and microphotographs were made with a Jeol 1200 EX (Japan) transmission electron 99

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Fig. 2. Scheme of isolation of cell wall fractions and their monosaccharide proportion (mol%) in primary and secondary phloem fibers of hemp stem.

monosaccharide yield was designated as the “lignin content”. The proportion between cellulose-retained and lignin-bound polysaccharides could vary depending on the duration of cellulase treatment. Thus, both fractions were considered together and designated as “strongly retained within cell-wall polysaccharides”. The fractionation procedure is summarized in Fig. 2. Cellulose content was calculated by subtracting the yields of all obtained fractions (buffer-extractable, AO-extractable, KOH-extractable, strongly retained within cell wall polysaccharides, and lignin)

from the dry mass of the initial sample. 2.4. Monosaccharide analysis Samples were hydrolyzed with 2 M TFA at 120 °C for 1 h, dried to remove TFA, dissolved in deionized water, and analyzed by high-performance anion-exchange chromatography using a DX-500 system equipped with a CarboPac PA-1 column (4 × 250 mm) and a pulsed amperometric detector ED40 (Dionex). The column temperature was 100

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30 °C, and the mobile phase (pumped at 1 mL min−1) consisted of 100% A (0–20 min), 90% A (20–21 min), 50% A (22–41 min), 0% A (42–55 min), and finally 100% A (56–85 min), where A was 16.5 mM NaOH and the other solvent (B) was 100 mM NaOH in 1 M sodium acetate. The results were analyzed using the PeakNet software according to the calibrations obtained for monosaccharide standards treated in advance with 2 M TFA at 120 °C for 1 h. The analysis was performed for two independent biological replicates and two analytical replicates of primary and secondary fibers. Results from this analysis in tables and diagrams are conveyed as means ± SD.

Table 2 Thickness of cell wall and its individual layers in the cross-section of primary and secondary phloem fibers of the middle part of hemp stem. Categories of fibers, according to cell wall thickness, μm

Thickness of secondary cell wall, μm

Primary fibers < 1.5 0.22 1.5–3.0 0.19 3.0–4.5 0.26 > 4.5 0.27 Secondary fibers < 1.5 0.48 0.47

2.5. NMR spectroscopy The structure of polymers extracted by buffer and obtained after the complete destruction of cellulose microfibrils from cell walls of primary and secondary hemp fibers was characterized by 1H NMR spectroscopy. The samples were dissolved in D2O (99.9%, Ferak, Germany) to accomplish the H-D exchange of hydroxyl protons in polysaccharides, dried and re-dissolved in D2O (99.994%, Aldrich, USA). NMR spectra were recorded on Bruker Avance III 600 MHz (Bruker, Germany) at 303 K. The residual HOD signal in all experiments was suppressed by means of presaturation. Data processing and spectra analysis were performed using Topspin 2.1 software (Bruker, Germany).

Thickness of tertiary cell wall layer, μm G

± ± ± ±

0.04 0.01 0.02 0.03

± 0.04 ± 0.03

0.58 1.61 2.59 4.40

Gn

± ± ± ±

0.13 0.19 0.12 0.45

– 0.40 ± 0.05

0.40 0.52 0.75 0.96

± ± ± ±

0.04 0.09 0.03 0.08

0.52 ± 0.09 0.47 ± 0.04

Total thickness of secondary and tertiary cell wall, μm

na

1.19 2.32 3.59 5.64

5 7 9 7

± ± ± ±

0.09 0.22 0.15 0.44

1.00 ± 0.11 1.34 ± 0.05

5 10

a Number of the analyzed photo taken from 3 plant stems; results are conveyed as means ± SD.

of the cell wall layer thickness upon deposition of additional cell wall material, we categorized the fibers according to the total cell wall thickness (Table 2). The cell wall layer located adjacent to the primary cell wall and middle lamellae, had the similar thickness in all analyzed primary fibers, irrespective of the total cell wall thickness. Same was true for secondary phloem fibers, in which the thickness of this cell wall layer was twice higher than in primary fibers ones, though the total cell wall thickness was considerably lower (Table 2). This cell wall layer of the fibers was effectively labeled with LM11 antibody specific for β-(1,4)-Dxylan (McCartney et al., 2005) (Fig. 4), while in the middle lamellae and primary cell wall as well as in the inner layers of thickened cell wall epitopes of the β-(1,4)-D-xylan were absent. In xylem cells, the secondary cell wall was uniformly labeled with LM11 through the whole thickness (data not shown). The outer layer of the thickened wall in hemp fibers was shown to contain the cellulose microfibrils with almost transverse orientation – over 80° (Dai and Fan, 2010). Together with the presence of xylan, these indicate that the layer is built as the common xylan-type secondary cell wall (Albersheim et al., 2010); thus, it was designated as a layer of the secondary cell wall of hemp fibers. The closest to plasma membrane newly formed cell wall layer looked “loosened” and “stripped”, both in primary and secondary phloem fibers of hemp stem (Fig. 3). Such picture was described also in the gelatinous cell wall of flax phloem fibers (Salnikov et al., 1993; Gorshkova et al., 2004, 2010). Between the secondary cell wall and the closest to plasma membrane “stripped” cell wall layer, there was the “solid” layer (Fig. 3), the thickness of which increased in parallel with total cell wall thickness (Table 2; Fig. 3E). In developed fibers, this layer becomes the major one. The thickness of the “stripped” cell wall layer did not vary as significantly as of “solid” one (Table 2; Fig. 3E). In mature fibers, the “stripped” cell wall layer was virtually absent. Both “solid” and “stripped” layers did not bind LM11 antibody but were labeled by INRA-RU2 antibody, specific for the RG-I backbone (Ralet et al., 2010), and N1 antibody, specific for flax β-galactosidase (Mokshina et al., 2012) (Fig. 4). Immunolabelling with LM5 antibody, specific for β-(1,4)-D-galactan (Jones et al., 1997), was not detected in both “solid” and “stripped” cell wall layers of hemp fibers (data not shown). Cell wall thickening in primary and secondary hemp fibers was accompanied by accumulation in cytoplasm of specific Golgi vesicles with characteristic bicolor (“dark-light”) appearance (Fig. 5A), similar to those described in flax fibers during the formation of gelatinous cell wall (Salnikov et al., 1993; Gorshkova et al., 2005). Sometimes we observed the detachment of “solid” layer from the secondary cell wall one (Fig. 5B), similar to how it happens in tension wood (Clair et al., 2005). Together with the absence of xylan (Fig. 4A, B) and lignin (Bonatti et al., 2004), quite low microfibril angle in the major cell wall

2.6. Immunodot analysis Total fractions containing 4, 1, and 0.25 μg of carbohydrates (2 μL) were applied to nitrocellulose membranes (0.2 μm; Sigma). Membranes were air dried for 30 min, washed for 5 min in PBST (phosphate-buffered saline with 0.05% Triton X-100), blocked for 1 h with phosphatebuffered saline containing 2% BSA, and then incubated for 40 min with the primary monoclonal antibody. The monoclonal antibodies used were LM2, LM5, LM6, LM11, LM13, LM15, LM19, LM21, JIM14 and INRA-RU2, all raised in rat except INRA-RU2 (mouse, hybridoma supernatant). LM2, LM5, LM6, LM11, LM13, LM15, LM19, LM21, and JIM14 were applied at 1:40 dilution and INRA-RU2 at 1:30 dilution, in PBST. After incubation with primary antibodies, membranes were washed three times for 10 min with PBST and then incubated with secondary biotinylated antibodies (Sigma; anti-rat to detect LM2, LM5, LM6, LM11, LM13, LM15, LM19, LM21 and JIM14 primary antibodies and anti-mouse to detect INRA-RU2) for 40 min. The membranes were washed again three times in PBST for 10 min, incubated for 30 min in streptavidin conjugated with alkaline phosphatase diluted 1:3000 and developed using a nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate kit (Silex). Potato galactan, linear arabinan from sugar beet, polygalacturonic acid from citrus pectin, xylan from beechwood, xyloglucan from tamarind seeds, galactomannan from carob (all supplied by Megazyme) and gum arabic from acacia tree (Sigma-Aldrich) were used as positive controls. 3. Results 3.1. Ultrastructural analysis and immunocytochemistry of phloem fiber cell wall in developing hemp stem Cell wall ultrastructure in the fibers of developing hemp stem was analyzed at the stage when the thickness of the cell wall in the primary phloem fibers varied from 1 to 6 μm (Table 2); in mature plants it reaches from 5 to 10 (Garcia-Jaldon et al., 1998) to 14 μm (Crônier et al., 2005). In the secondary phloem fibers, which are known to have several-fold smaller cell diameter than the primary ones (Snegireva et al., 2015), the cell wall thickness was below 2 μm. The structure of the fiber cell wall in developing hemp stem was not uniform: three layers, besides the middle lamellae and the primary cell wall, were well distinguished without any additional labeling both in the primary and secondary phloem fibers (Fig. 3). To follow the changes 101

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Fig. 3. Deposition and remodeling of the tertiary cell wall in phloem fibers of hemp stem. Early developmental (A) and late developmental (B) stages of primary fiber cell wall. Early developmental (C) and late developmental (D) stages of secondary fiber cell wall. Scheme of sequential steps in deposition and remodeling of tertiary cell wall layers in both primary and secondary hemp fibers (E). G and Gn – tertiary cell wall layers, ML – middle lamellae, PCW – primary cell wall, S – secondary cell wall, SP – simple pore; scale bar 1 μm.

layer takes place both in primary and secondary phloem fibers of hemp (Fig. 3E), similar to the flax fibers (Gorshkova et al., 2004; Gorshkova and Morvan, 2006; Mikshina et al., 2013).

layer of hemp fibers (Dai and Fan, 2010), all the above indicates that the “solid” and “stripped” layers of hemp fiber cell wall correspond, respectively, to G and Gn (from “gelatinous” and “gelatinous-new”) layers of tertiary cell wall (Mikshina et al., 2013). Increase of the Glayer thickness through the development of cell wall suggests that postdeposition remodeling of the “stripped” Gn-layer into the “solid” G-

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Fig. 4. Immunolocalization of cell wall polysaccharides and β-galactosidase in phloem fibers of hemp stem. Immunolabelling of primary (left) and secondary (right) fibers with (A, B) LM11 antibody specific to β-(1,4)-D-xylan, (C, D) INRA-RU2 antibody specific to RG-I backbone, (E, F) N1 antibody specific for flax fiber β-galactosidase. Silver enhancement was applied for different times (2–5 min), leading to different sizes of particles. G and Gn – tertiary cell wall layers, ML – middle lamellae, PCW – primary cell wall, S – secondary cell wall; scale bar 1 μm.

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Fig. 5. Ultrastructure of phloem fibers of hemp stem at the stage of tertiary cell wall deposition. (A) Specific Golgi vesicles in the cytoplasm of primary fibers during the formation of the tertiary cell wall. (B) A dtachment of “solid” cell wall layer (shown by arrowhead) from the secondary one in primary fibers. ER – endoplasmic reticulum, G, and Gn – tertiary cell wall layers, GA – Golgi apparatus, GV – Golgi vesicles, ML – middle lamellae, PCW – primary cell wall, S – secondary cell wall; scale bar 1 μm.

3.2. Cell wall composition of hemp fibers

(Table 3, Fig. 2). The composition of polysaccharides extracted by AO and KOH from primary and secondary fiber cell walls was similar to each other and to that described by D. Crônier et al. (2005) for fiberenriched samples of hemp stem, except that in pectic fraction of isolated fibers the content of galacturonic acid was higher and that of arabinose – lower. Xylose-, mannose- and glucose-containing polymers prevailed in the KOH-extractable fraction, some of these polymers were released also by AO; however, the main component of the latter was pectin polysaccharides, consisting of galacturonic acid, rhamnose, galactose and arabinose (Fig. 2).

In general, yield and total composition of the cell wall polymers of isolated primary and secondary phloem fibers were similar (Table 3). The overwhelming cell wall component of both types of fibers was cellulose, which comprised 89% of dry weight of cell wall (Table 3). Glucose, part of which belongs to amorphous cellulose, xylose, and galactose, predominated in the composition of TFA-hydrolysable polysaccharides. The increased portion of xylose in secondary fibers corresponds to the thicker xylan-rich S-layer in them (Tables 2 and 3, Fig. 4F). Non-cellulosic polymers comprised 3–4% of cell wall dry weight as determined by the total yields of OA- and KOH-extraсtable fractions and polymers strongly retained within cell wall (without glucose)

3.2.1. Buffer-extractable polymers Galactose, galacturonic acid, rhamnose, and arabinose were the main monosaccharides present in the polymers of buffer-extractable

Table 3 Yields of cell wall fractions and monosaccharide composition of TFA-hydrolysable polymers from the cell wall of isolated primary and secondary hemp fibersa. Type of fibers

Primary fibers Secondary fibers

Cell wall fractions, % of dry weight of cell wall Buffer-extractableb

AO-extraсtableb

KOH-extraсtableb

Strongly retained within cell wallc

Cellulosed

Lignine

2.3 ± 1.0 1.9 ± 0.1

0.6 ± 0.1 0.9 ± 0.6

2.1 ± 0.2 2.6 ± 1.0

1.3 ± 0.4 1.1 ± 0.4

89.4 ± 0.2 88.9 ± 1.7

4.5 ± 0.1 4.6 ± 0.2

Cell wall monosaccharide proportion, mol%

Primary fibers Secondary fibers

Rha

Ara

Gal

Glc

Xyl + Man

GalA

GlcA

2.5 ± 0.7 3.7 ± 0.6

1.4 ± 0.4 2.1 ± 0.4

11.9 ± 0.4 12.1 ± 0.0

67.3 ± 4.4 55.7 ± 2.6

11.1 ± 1.4 19.6 ± 0.2

4.7 ± 2.2 6.0 ± 1.0

1.1 ± 0.2 0.8 ± 0.4

Cell wall monosaccharide composition, mg/g of cell wall dry weight

Primary fibers Secondary fibers

Rha

Ara

Gal

Glc

Xyl + Man

GalA

GlcA

2.9 ± 1.6 5.1 ± 1.3

1.6 ± 0.9 2.6 ± 0.8

15.3 ± 2.1 17.9 ± 0.9

86.4 ± 7.9 82.2 ± 1.6

13.4 ± 4.8 26.6 ± 1.6

7.0 ± 5.5 9.6 ± 2.8

1.6 ± 0.7 1.3 ± 0.9

a

Data are presented as means ± SD. Determined as the sum of yields of all monosaccharides obtained by TFA hydrolysis from the corresponding fraction. c Determined as the sum of yields of all monosaccharides obtained by TFA hydrolysis from the fraction of polymers, tightly retained by cellulose and lignin. d Calculated from the dry mass of the initial sample by subtracting yields of buffer-extractable, AO-extractable, KOH-extractable, polymers, tightly retained by cellulose and lignin and lignin. e Calculated from the dry mass of the pellet remaining after cellulose digestion by subtracting yields of monosaccharide obtained by TFA hydrolysis from this pellet. b

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Fig. 6. Biochemical analysis of buffer-extractable polymers from phloem fibers of hemp stem. 1H NMR spectra, immunodot binding assays and iodine test of buffer-extractable polymers obtained from (A) primary and (B) secondary hemp fibers.

arabinan – was extremely weak. The results of immunodot analysis were confirmed by NMR data. Anomeric regions of 1Н NMR spectra of total fractions of buffer-extractable polymers included the intensive signals ascribed to H1 1,2and 1,2,4-Rha (5.24 ppm) and H1 1,4-GalA (5.03–5.11 ppm) belonging to RG-I backbone (Van Hazendonk et al., 1996; Mikshina et al., 2012) as well as the signals at 4.48 ppm and 4.54 ppm (Fig. 6) referring to 1,6Gal and 1,3,6-Gal of AG-II (Golovchenko et al., 2007; Shakhmatov

fractions of both types of hemp fibers (Fig. 2). Immunolabeling of buffer-extractable fractions by RU-2 antibody suggested the presence of rhamnogalacturonan I (RG-I). The binding of LM5 revealed the presence of β-(1,4)-galactan in fractions of both fiber types, although labeling with this antibody was rather weak (Fig. 6). Binding of LM2 and JIM14 antibody indicated the presence of arabinogalactans (Fig. 6). Application of LM6 indicated the presence of α-(1,5)-arabinan, however, labeling by LM13–another antibody specific for linear α-(1,5)105

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extractable polysaccharides (Fig. 6) are associated with the disappearance of AGP signals, as well as the appearance of the signals from β-1,4-Glc, β-1,4-Xyl and β-1,4-Man protons, the intensive signals of H1 in the region of 4.49–4.52 ppm and 4.77–4.90 ppm, as well as H2–at 3.30–3.41 ppm and 4.11–4.13 ppm among them (York et al., 1990; Hannuksela and Hervé du Penhoat, 2004; Shakhmatov et al., 2014). Weak binding of LM15 and LM11 antibodies of polymers released after cellulose degradation (Fig. 7) suggested the presence of trace amounts of xyloglucan and xylan in their composition. 1 H NMR spectrum of polysaccharides strongly retained within cell wall (Fig. 7) confirmed the presence of RG-I (signals belonging to H1 Rha (5.24 ppm), H1 GalA (including GalA attached to Rha residue in the region of a stronger field (5.03–5.05 ppm) in comparison with GalA of PGA (5.09–5.11 ppm)) and CH3 groups of 1,2-Rha (1.24 ppm) and 1,2,4-Rha (1.31 ppm)) and β-1,4-linked Gal (characteristic signals of H1 at 4.60–4.64 ppm and H4 at 4.16 ppm (Davis et al., 1990; Gurjanov et al., 2008; Mikshina et al., 2012)). To further characterize the structure of the polymer, the proportion of unbranched Rha and Rha substituted by side chains of different length, as well as the average length of oligomeric side chains, were calculated according to the earlier stepby-step description (Mikshina et al., 2012). The proportion of branched and unbranched Rha residues in the polysaccharides released after cellulose degradation was 52% and 48%, respectively as calculated from the ratio of signals at 1.24 and 1.31 ppm. The proportion of Rha branched with a single Gal residue was considered as equal to the proportion of terminal Gal that was calculated from the integral

et al., 2014, 2015). Signals at 2.7–3.2 ppm and in the regions 0.7–2.5 ppm (Fig. 6) from functional groups of amino acids in the 1Н NMR spectra and absorption at 230 nm belonged to the peptide bond (data not shown) indicated the presence of proteins, which can be a component of AGP. The presence of signals at 5.40 ppm and in the region of 3.67–3.83 ppm (Fig. 6A) belonging to H1 and H5, H6 of α-Glc (Seefeldt et al., 2008) in the spectrum of buffer-extractable polymers from primary fibers explained the increased proportion of glucose in this type of fiber (Fig. 2) by the presence of starch, which was also confirmed by a positive iodine test (Fig. 6A). 3.2.2. Polymers strongly retained within the cell wall The proportion of polysaccharides that remained in cell wall residue after AO- and KOH-extractions was similar in primary and secondary hemp fibers and accounted about 1% of dry weight of cell wall (Table 3). Galacturonic acid, xylose (mannose), glucose, as well as galactose and rhamnose constituted the bulk of polymers that were strongly retained within the cell wall, in both types of fibers (Fig. 2). The key features of polymers released after cellulose degradation were the intensive binding of RU-2 and LM5 specific to RG-I backbone and to tetramer of β-(1,4)-Gal, respectively, as well as the extremely low intensity of 1,5-arabinan labeling by LM6 and LM13 and the absence of labeling by antibodies specific to AGP (LM2 and JIM 14) (Fig. 7). Indeed, a number of changes in the 1H NMR spectra of polymers released after cellulose degradation (Fig. 7) in comparison with buffer-

Fig. 7. Biochemical analysis of polymers strongly retained within cell wall in phloem fibers of hemp stem. 1H NMR spectrum and immunodot binding assays of strongly retained by cellulose polymers in secondary hemp fibers.

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xylan-enriched layer is the layer of the secondary cell wall. The thickness of the secondary cell wall is among the most obvious differences between gelatinous fibers from different plant species. In hemp fibers, the xylan layer is well developed and can be distinguished by transmission electron microscopy even without additional staining (Fig. 3). In flax, it is not possible to see the distinct layer, but the LM11 antibody reveals the epitope in the layer located outside the tertiary (gelatinous) cell wall (Gorshkova et al., 2010). In tension wood fibers, G-layer is always made after at least S1 (or sometimes S1 + S2, or even S1 + S2 + S3) layer of xylan type (Mellerowicz et al., 2001; Sultana and Rahman, 2013; Ghislain and Clair, 2017). So, within the cell wall of gelatinous fibers from different plant species, the formation of the tertiary (gelatinous) cell wall is always preceded by the deposition of the xylan one. We can assume that the deposition of xylan cell wall is necessary to fix the fiber radial size since the axially oriented microfibrils of gelatinous cell wall cannot prevent changes in fiber width. The difference in the thickness of the secondary cell wall in the primary and secondary fibers (Table 2) can be related to the difference in their lignification. In the secondary hemp fibers, the xylan cell wall is wider and the lignification degree higher than in the primary ones. In gelatinous fibers lignin content is low and the polymer is deposited mainly in the peripheral regions of the cell wall, including middle lamellae, primary cell wall and secondary cell wall (Bonatti et al., 2004; Fernandez-Tendero et al., 2017). Lignification occurs mainly at the late stages of fiber development (Crônier et al., 2005). To get to the outer part of the cell wall at this stage of fiber development the monolignols have to pass through the thick gelatinous cell wall to be polymerized only in the cell wall periphery. The question is what prevents monolignols from polymerization within the gelatinous layer? One of the possible answers is the distribution of peroxidases or laccases, which are necessary to initiate assembly of monolignols into the polymer. However, the difference in matrix polysaccharides between the layers with so different lignification degree suggests some role of these polymers in lignification process. All this makes gelatinous fibers an interesting model to study the regulation of lignin formation.

intensity of the well-separated H2 signal (3.51 ppm); it accounted 37–42% of the total Rha residues. So, the proportion of Rha substituted by oligomeric galactan side chains (obtained by subtraction of the summarized content of unsubstituted Rha and Rha substituted with the single galactose from the total content of Rha) comprised 10–15%. The average length of these side chains was not more than 7–10 residues if one considered that all β-1,4-Gal belonged to the RG-I side chains. Calculation of this parameter was based on the ratio of integral intensities of Gal signal (H1, 4.64 ppm) and signals from the three protons of the methyl group of 1,2,4-Rha. The possible underrating of Gal/Rha ratio at the analysis of signal integral intensity by NMR was corrected according to the monosaccharide composition. Thus, the average length of oligomeric side chains of RG-I strongly retained within the cell wall of hemp fibers is lower than for the similar polymer from flax fibers (Mikshina et al., 2012). This can explain the less intensive labeling of polysaccharides of this cell wall fraction of hemp fibers by LM5 antibody. At the same time, these data demonstrate a significant similarity of polysaccharides strongly retained within the cell wall of hemp and flax phloem fibers. 4. Discussion 4.1. Tertiary cell wall layers in hemp fibers Transmission electron microscopy reveals well distinguishable layers in cell walls of primary and secondary phloem fibers of hemp, with the general arrangement being similar in both fiber types. The first layer of the thickened cell wall that is adjusted to the primary cell wall and is deposited right after the end of cell elongation is identified as the xylan-rich layer, typical for secondary cell walls of angiosperms. The major part of the thickened cell wall in hemp phloem fibers is very different from that and can be referred to as tertiary cell wall (also named gelatinous or G-layer) described for flax (Gorshkova et al., 2010; Mikshina et al., 2013) and for tension wood (Gorshkova et al., 2015; Guedes et al., 2017). The key features of the tertiary cell walls are the axial orientation of microfibrils, absence of xylan and lignin (at least at the stage of deposition), the presence of RG-I and important processes of cell wall maturation. In hemp, same as in flax (Salnikov et al., 1993; Gorshkova et al., 2004, 2010; Gorshkova and Morvan, 2006; Mikshina et al., 2013), the formation of tertiary cell wall in primary and secondary phloem fibers begins with the deposition of the “stripped” Gn-layer (from “gelatinousnew”). During further cell wall development “stripped” layer is transformed to “solid” G-layer (from “gelatinous”), as a result, the thickness of the G-layer increases. In mature fibers Gn-layer disappears, being completely transformed into G-layer. It should be noted that the thickness of primary and secondary cell wall layers remains the same in the course of tertiary cell wall deposition (Fig. 3, Table 2). Such post-synthetic modification of the tertiary cell wall layers is associated with the decrease in the length of β-(1,4)-D-galactan side chains of rhamnogalacturonan I (Mikshina et al., 2013) and release of free galactose, which occurs with the participation of fiber-specific βgalactosidase (Roach et al., 2011; Mokshina et al., 2012). Also, during the transformation of the tertiary cell wall layers the crystallinity of cellulose is increased (Gorshkova and Morvan, 2006). The distribution of epitopes of the antibody N1, specific for β-galactosidase of flax fibers (Mokshina et al., 2012) was also observed in both layers of the tertiary cell wall of hemp fibers (Fig. 4).

4.3. Development of primary and secondary phloem fibers in hemp stem, compared to that of flax fibers The obtained results permit to compare the development of two types of phloem fibers that have the same genetic background and also while referring to flax fibers – to find general regularities and peculiarities in the formation of gelatinous phloem fibers in phylogenetically distinct higher plant species. The major similarities in the development of the flax primary phloem fibers (Gorshkova et al., 2003, 2004, 2005) and of the hemp primary and secondary phloem fibers originating from different meristems, include the following: 1. Deposition of tertiary (gelatinous) cell wall is accompanied by accumulation of the tissue- and the stage-specific polymer that has rhamnogalacturonan I backbone. It can be collected from tissue homogenization buffer, being not yet fixed within the cell wall, in samples of developing phloem fibers of flax and hemp, both primary and secondary ones in the latter case. In flax, side chains composed mainly of β-(1,4)-D-galactan are attached to RG-I (Mikshina et al., 2012) and are important for the self-association of RG-I molecules that has functional significance (Mikshina et al., 2017; Makshakova et al., 2017). In hemp, the simultaneous presence of RG-I backbone and β-(1,4)-D-galactan indicates the same structure; the linkage between the backbone and side chains was proved (Vignon and Garcia-Jaldon, 1996). Similar polysaccharide(s) are strongly retained by cellulose microfibrils and remain unextracted after treatment with concentrated alkali, both in flax and hemp phloem fibers. 2. The newly deposited layers of gelatinous cell wall undergo significant remodeling that leads to transformation of Gn into G-layer. The course of such transformation looks quite similar in phloem

4.2. Xylan layer in gelatinous fibers Xylan epitopes in the outer cell wall part of hemp fibers were found earlier by fluorescent microscopy; however, they were ascribed to the primary cell wall (Blake et al., 2008). The electron microscopy of the intracellular junction between fibers with thickened cell wall and nonfiber cell with primary cell wall (Fig. 3B) clearly demonstrates that 107

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fibers of flax and hemp and may involve post-synthetic modification of the β-(1,4)-D-galactan. Antibody N1 raised against flax fiberspecific β-galactosidase also binds effectively tertiary cell walls in both types of hemp phloem fibers. 3. Formation of the tertiary (gelatinous) cell wall always occurs subsequent to the deposition of secondary (xylan) cell wall layer.

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Golovchenko, V.V., Bushneva, O.A., Ovodova, R.G., Shashkov, A.S., Chizhov, A.O., Ovodov, Yu.S., 2007. Structural study of bergenan, a pectin from Bergenia crassifolia. Rus. J. Bioorg. Chem. 33 (1), 47–56. http://dx.doi.org/10.1134/ S1068162007010050. Gorshkova, T., Morvan, C., 2006. Secondary cell-wall assembly in flax phloem fibres: role of galactans. Planta 223 (2), 149–158. http://dx.doi.org/10.1007/s00425-0050118-7. Gorshkova, T.A., Wyatt, S.E., Salnikov, V.V., Gibeaut, D.M., Ibragimov, M.R., Lozovaya, V.V., Carpita, N.C., 1996. Cell-wall polysaccharides of developing flax plants. Plant Physiol. 110 (3), 721–729. http://dx.doi.org/10.1104/pp.110.3.721. Gorshkova, T.A., Salnikov, V.V., Chemikosova, S.B., Ageeva, M.V., Pavlencheva, N.V., van Dam, J.E.G., 2003. Snap point: a transient point in Linum usitatissimum bast fiber development. Ind. Crops Prod. 18, 213–221. http://dx.doi.org/10.1016/S09266690(03)00043-8. 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The differences in the development of the studied gelatinous fibers include: a) The thickness of the secondary cell wall layer of xylan type varies from barely determined (in flax) to significant. In all analyzed phloem fibers, only one layer of the secondary cell wall is deposited, while in wood fibers, up to three such layers with different microfibril orientation can be observed (Mellerowicz et al., 2001; Sultana and Rahman, 2013; Ghislain and Clair, 2017). b) The length and the diameter of individual cells differ significantly between primary and secondary fibers. This is largely due to the effectiveness of intrusive elongation, which is higher for primary fibers than for secondary ones (Snegireva et al., 2015). c) The structure of the tissue-specific RG-I may differ. In flax fibers, the proportion of GalA and Rha are similar and the sequences of consecutive GalA residues are absent (Mikshina et al., 2017) indicating that the polymer is pure RG-I. In hemp, the higher proportion of GalA as compared to Rha (Table 3), same as labeling by LM19 antibody indicates the possibility of homogalacturonan chains. However, whether RG-I and HG are present as the parts of the same molecule or as a mixture of different polymers still has to be checked. The length of β-(1,4)-galactan chains in the entrapped by cellulose microfibrils’ polymers are longer in flax than in hemp. Backbone of RGs-I from hemp and tension wood fibers has the same proportion of branched rhamnose – 52 and 53% of all rhamnose content, correspondingly (Gorshkova et al., 2015); backbone of flax RG-I is more substituted by side chains and contains 72% of 2,4-Rha (Mikshina et al., 2012). Hemp fibers are characterized by the smallest average length of galactan chains strongly retained by cellulose microfibrils − 7–10 residues; this parameter is the maximum for RG-I from tension wood fibers (18 residues) and takes an intermediate value (14 residues) for the flax RG-I. Thus, the obtained results show the basic similarities in the biogenesis of gelatinous phloem fibers, making the regularities, previously found for flax, more general. The developed approaches can be used to analyze the formation of other plant fibers to give the basis for their classification and to find the biological determinants of fiber quality parameters. Acknowledgments The work was supported by the Russian Science Foundation, project 16-14-10256 (TG, NI, and TC; microscopy and immunochemistry of Gfibers, immunodot analysis) and the Russian Foundation for Basic Research, projects 15-04-02560 (PM and OS; analysis of rhamnogalacturonan I structure by NMR spectroscopy) and 15-04-05721 (TC; isolation of cell wall polysaccharide fractions and their monosaccharide analysis). We thank Prof. J. Paul Knox (University of Leeds, UK) and Dr. Fabienne Guillon (Institut National de la Recherche Agronomique, France) for providing the antibodies as well as Dr. G. S. Stepanov and Dr. I. V. Romanova (Chuvash Research Institute of Agriculture, Tsivilsk, Russia) for growing the hemp plants. References Albersheim, P., Darvill, A., Roberts, K., Sederoff, R., Staehelin, A., 2010. Plant cell walls. Garland Science. Arend, M., 2008. Immunolocalization of (1, 4)-β-galactan in tension wood fibers of

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