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First published online April 7, 2006; 10.1104/pp.106.077701 Plant Physiology 141:651-662 (2006) © 2006 American Society of Plant Biologists Response of the Leaf Cell Wall to Desiccation in the Resurrection Plant Myrothamnus flabellifolius1Department of Molecular and Cellular Biology, University of Cape Town, Rondebosch 7701, South Africa (J.P.M., G.G.L., W.F.B., J.M.F.); and Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6037, Institut Fédératif de Recherche Multidisciplinaire sur les Peptides 23, Centre Commun de Microscopie Electronique, Université de Rouen, 76821 Mont Saint Aignan cedex, France (E.N.-O., L.C., P.L., A.D.)
The Myrothamnus flabellifolius leaf cell wall and its response to desiccation were investigated using electron microscopic, biochemical, and immunocytochemical techniques. Electron microscopy revealed desiccation-induced cell wall folding in the majority of mesophyll and epidermal cells. Thick-walled vascular tissue and sclerenchymous ribs did not fold and supported the surrounding tissue, thereby limiting the extent of leaf shrinkage and allowing leaf morphology to be rapidly regained upon rehydration. Isolated cell walls from hydrated and desiccated M. flabellifolius leaves were fractionated into their constituent polymers and the resulting fractions were analyzed for monosaccharide content. Significant differences between hydrated and desiccated states were observed in the water-soluble buffer extract, pectin fractions, and the arabinogalactan protein-rich extract. A marked increase in galacturonic acid was found in the alkali-insoluble pectic fraction. Xyloglucan structure was analyzed and shown to be of the standard dicotyledonous pattern. Immunocytochemical analysis determined the cellular location of the various epitopes associated with cell wall components, including pectin, xyloglucan, and arabinogalactan proteins, in hydrated and desiccated leaf tissue. The most striking observation was a constitutively present high concentration of arabinose, which was associated with pectin, presumably in the form of arabinan polymers. We propose that the arabinan-rich leaf cell wall of M. flabellifolius possesses the necessary structural properties to be able to undergo repeated periods of desiccation and rehydration.
Desiccation tolerance of vegetative plant tissue is a phenomenon found throughout the plant kingdom (Bewley and Krochko, 1982  ). It is more common among lower plants, such as mosses and ferns, whereas it is rarely observed in angiosperm families (Alpert and Oliver, 2002). Plants that display desiccation tolerance are commonly called resurrection plants (Oliver, 1996), so named because of their ability to lose most of their cellular water (down to <5% relative water content [RWC]), cease metabolic function, and exist in a quiescent, desiccated state for extended periods and then rehydrate their vegetative tissue and resume metabolism (Gaff, 1977). The largest resurrection plant is Myrothamnus flabellifolius, a woody shrub distributed throughout central southern Africa (Sherwin et al., 1998; Glen et al., 1999). The family Myrothamnaceae, which contains only two species, M. flabellifolius and Myrothamnus moschatus (Glen et al., 1999), diverged from the Gunnera, one of the oldest surviving angiosperm genera, approximately 1.2 x 108 years ago and is therefore likely to have been one of the earliest angiosperm families to have acquired desiccation tolerance (Wanntorp et al., 2001). Mechanisms proposed to explain the ability of resurrection plants to survive desiccation include Suc and trehalose accumulation (Bianchi et al., 1993; Drennan et al., 1993), accumulation of stress proteins (Goyal et al., 2005; Mtwisha et al., 2006), as well as the presence of polyphenols, in particular galloylquinic acids, which have been shown to act as antioxidants and membrane protectants (Moore et al., 2005). These mechanisms have evolved to counteract stresses imposed during desiccation and subsequent rehydration (Alpert and Oliver, 2002). The most visible result of desiccation is a considerable reduction in tissue and cell volume that occurs due to water loss (Farrant, 2000). Thus, the cell volume of Craterostigma wilmsii has been reported to be on the order of 78% less upon desiccation (Farrant, 2000) and is accompanied by a concomitant withdrawal of the plasma membrane from the cell wall as well as extensive cell wall folding (Vicré et al., 2004b) believed to result in mechanical stress (Farrant, 2000; Vicré et al., 2004a). Mechanical stress is defined as the tension that develops between and within the plasma membrane and the cell wall due to a loss of turgor pressure, resulting in plasmolysis (Walters et al., 2002). The damage resulting from mechanical stress is dependent on factors such as the strength of plasmalemma-cell wall contacts, degree of plasma membrane disruption, as well as elasticity of the cell wall (Walters et al., 2002). It has been proposed (Iljin, 1957) that plants must overcome such mechanical stress to acquire desiccation tolerance. Strategies employed by desiccation-tolerant organisms to counteract mechanical stress include storage within vacuoles of proteins, lipids, and carbohydrates to replace lost water and cell wall folding to prevent possible rupture of the plasma membrane (Vicré et al., 2004a). Desiccation-induced wall folding has been proposed (Webb and Arnott, 1982) to be essential for structural preservation of tissue and the extent and manner of such folding is species specific and dependent upon the chemical composition and molecular architecture of the cell wall.
Plant cell walls are dynamic entities that govern the morphology, growth, and development of plants (Albersheim et al., 1994
The pronounced morphological changes observed on desiccation of M. flabellifolius are similar to those observed in other resurrection plants undergoing the same process (Alpert and Oliver, 2002 ; Vicré et al., 2004a). Dehydrating M. flabellifolius plants undergo curling of both their stem segments as well as their leaves (Fig. 1
). In particular, the leaves fold upward to a position where the adaxial surfaces are appressed against the stem (Fig. 1) and the sclerenchymous leaf ribs (Grundell, 1933) move closer to one another, thereby closing the leaf face, which has been proposed to minimize the exposure of the desiccated leaf to light (Farrant et al., 2003). Electron microscopy of hydrated and desiccated leaves revealed further morphological changes that occurred upon desiccation (Fig. 2, A–H
). Cryoscanning electron microscopy of the epidermal surface of hydrated leaves revealed a distinctive network pattern of stomata and gland cells interdispersed between regular epidermal cells (Fig. 2A). Upon desiccation, these epidermal cells underwent massive cell wall folding and shrinkage around the seemingly less flexible stomata and gland cells (Fig. 2B). The stomata in particular were clearly visible in the desiccated state, displaying little of the extreme morphological changes present in desiccated epidermal cells (Fig. 2B). Freeze-fracture cross sections through cryofixed leaves showed that considerable changes in tissue and cellular structure occurred associated with the desiccation process (Fig. 2, C–F). Electron microscopy of a cross section through a frozen hydrated leaf (Fig. 2, C and E) revealed clear anatomical features, such as the epidermis, spongy mesophyll layer, palisade mesophyll layer, as well as vascular cells. In contrast, electron microscopy of a cross section through an identically treated desiccated leaf showed no discernible anatomical organization (Fig. 2, D and F). It would appear that cell wall folding had occurred during desiccation, resulting in a compacted, wrinkled appearance of the leaf cell layers. Only sclerenchymous and vascular cells resistant to folding, as well as epidermal cells, were clearly identifiable (Fig. 2, D and F). Higher magnification examination of hydrated tissue (Fig. 2E) revealed turgid endodermal cells and small (approximately 30-µm diameter) spongy mesophyll cells with multiple plasmodesmata connections to adjacent cells. Several intercellular airspaces and calcium oxalate druse crystals (Grundell, 1933) were also clearly visible. Desiccated leaf tissue at the same magnification (Fig. 2F) showed none of the features evident in hydrated tissue. Only folded cell wall fragments, most likely the result of mechanical fracturing during sample preparation, a greater number of intercellular airspaces, and amorphous deposits between cell wall layers, were observed. These amorphous deposits were possibly the result of desiccation of salts and cytosolic constituents.
Examination of chemically fixed hydrated and desiccated leaf tissue by transmission electron microscopy provided further evidence for the changes observed using scanning electron microscopy (Fig. 2, G and H). Transverse sections of hydrated leaves (Fig. 2G) showed turgid mesophyll cells typical of turgid tissue, with distinct cell walls, plasmodesmata, cytoplasm, and associated constituents such as chloroplasts, starch granules, and large central vacuoles. The cytoplasm of these cells occurred on the periphery of the cells adjacent to the cell wall. In contrast, transverse sections of desiccated leaves (Fig. 2H) showed cells with folded cell walls and a compact dense cytoplasm separated from the cell wall in a manner that resembled that observed in plasmolyzed plant cells, although the plasmalemma remained intact. The plasmolyzed appearance of desiccated cells might have been brought about by use of an aqueous aldehyde-based fixative (Hayat, 1981 ). This would preferentially fix cytoplasmic proteins rather than the cell wall carbohydrates because the former have a higher number of amino groups (Hayat, 1981). Because desiccated cell walls would presumably have swelled during the fixation process, our observations of cell wall folding in fixed desiccated cells provide strong evidence that cell wall folding occurs in vivo, and this is probably the main mechanism of mechanical stabilization of the mesophyll cells of this species.
Because the cell wall of M. flabellifolius might be unique on account of its taxonomic position (Qiu et al., 1998
We next fractionated the total cell walls prepared from hydrated and desiccated M. flabellifolius leaves (Table I ; Fig. 4 ) by serial extraction, initially with phosphate buffer at 80°C, then with CDTA, and finally with increasing concentrations of KOH. These latter extractions were all performed at 20°C. Each fraction extracted (A–I; Table I) was gravimetrically analyzed before being hydrolyzed with TFA; insoluble material remaining after TFA hydrolysis of fraction I was hydrolyzed with H2SO4 (fraction J). These hydrolysates were then analyzed for the presence of individual monosaccharides (Fig. 5 ). Gravimetric analysis of lyophilized fractions recovered from serial fractionation (Table I) yielded three significant differences in fractions B, D, and E between hydrated and desiccated leaves. CDTA extraction (fraction B) extracted more material from hydrated (10.9% ± 0.4%) than from desiccated (8.0% ± 0.5%) samples; more 0.05 M KOH insoluble material (fraction D) was present in desiccated (12.3% ± 1.5%) compared with hydrated (9.7% ± 0.4%) samples and 1 M KOH (fraction E) extracted almost double the material from hydrated (8.2% ± 1.6%) than from desiccated (4.1% ± 0.6%) samples.
The monosaccharide composition of each fraction obtained after cell wall fractionation was determined (Fig. 5), allowing the ability to infer the main polymers present. Fraction A contained predominantly Ara, GalUA, Man, and Gal, consistent with mannoproteins, arabinogalactan proteins (AGPs), soluble pectin, and other glycoproteins being extracted with phosphate buffer at 80°C. There were significant differences in the levels of GalUA, Gal, and Ara between hydrated and desiccated leaves, with hydrated leaves found to contain increased levels of GalUA and decreased levels of Gal and Ara. Fraction B contained predominantly Ara and GalUA, consistent with CDTA-mediated solubilization of pectin. The presence of Ara suggested the association of neutral arabinan chains together with the pectin, in agreement with previous data presented (Fig. 3), which revealed high Ara and GalUA content in the cell wall. Furthermore, less Ara was present in hydrated compared with desiccated leaves. Fraction C contained Ara, GalUA, Glc, Rha, and Xyl as the major monosaccharides present. Of these monosaccharides, relatively increased amounts of Xyl and Glc and a decreased amount of GalUA were found to be present in hydrated leaves. Fraction D contained Ara, GalUA, and Rha as the most abundant monosaccharides present, with increased levels of Ara and Rha and a decreased level of GalUA found in hydrated leaves. The high amounts of Ara and GalUA present in these fractions (C and D) indicated that additional pectic material is present in desiccated leaves associated with arabinans tightly bound to the cell wall and only released by strong alkali extraction. Rhamnogalacturonan polymers were also inferred to be present due to the presence of Rha in both extracts. Fraction E contained primarily Xyl, with lesser amounts of Ara and Glc, together with small amounts of the other monosaccharides, consistent with the presence of arabinoxylans in this fraction. Hydrated leaves were found to contain increased Xyl and Glc and decreased Ara concentrations. Fraction F contained almost exclusively Xyl, with no significant difference in concentration between the hydrated and desiccated states. The high concentration of Xyl in both the soluble and insoluble 1 M KOH extracts (fractions E and F) suggested the presence of the more soluble arabinoxylans in fraction E and the insoluble crystalline xylan polymers in fraction F. Continued serial extraction with 4 M KOH and subsequent TFA hydrolysis failed to reveal any significant differences between hydrated and desiccated leaves. Material extracted with 4 M KOH (fraction G) contained mainly Xyl and Glc, together with lesser amounts of Ara, Man, and Gal, consistent with the presence of xyloglucan polymers. Insoluble material after extraction with 4 M KOH (fraction H) contained chiefly Xyl, suggesting the presence of residual insoluble crystalline xylans. The alkali-insoluble residue remaining after serial extraction was finally subjected to successive acid hydrolysis using first TFA (fraction I) and then H2SO4 (fraction J). Fraction I was found to contain high amounts of Ara, together with lesser amounts of GalUA and Xyl, suggesting the presence of residual tightly bound pectic arabinans and xylans. Fraction J contained mostly Glc, most likely derived from crystalline cellulose. Mannans have been reported to be associated with cellulose (Fry, 1988 ); the presence of Man in this fraction suggested such an association in M. flabellifolius leaves.
To characterize the xyloglucan component of M. flabellifolius leaf hemicellulose, the major load-bearing polymers of the plant cell wall, isolated cell walls, were subjected to enzymatic degradation and analysis. Released oligosaccharides were identified by HPLC (data not shown) with further confirmation by matrix-assisted laser-desorption ionization (MALDI)-time-of-flight (TOF) mass spectrometry. Enzymatic degradation resulted in the release of four predominant xyloglucan-derived oligosaccharides with mass-to-charge ratio (m/z) 1,084, 1,288, 1,435, and 1,639 (Fig. 6
). The m/z 1,084 ion was assigned to XXXG (nomenclature according to Fry et al. [1993]
AGPs have been reported to be associated with the cell wall and plasma membrane of plant cells (Knox, 1997 ), where it has been proposed that they function in a structural capacity likely to facilitate important processes in plant growth and development (Showalter, 2001). A study of the monosaccharide content of AGPs isolated using the Yariv reagent (Schultz et al., 2000) from hydrated and desiccated M. flabellifolius leaf tissue was performed. This showed an abundance of Ara, Gal, GalUA, and Man from both hydrated and desiccated leaves with elevated levels of GalUA and Gal observed in hydrated samples (Fig. 7
). In addition, quantification of Yariv-precipitable AGPs using rocket electrophoresis did not reveal any difference between hydrated and desiccated samples (data not shown).
The distribution of various carbohydrate epitopes present in or near the cell walls of hydrated and desiccated M. flabellifolius leaves was next investigated using immunocytochemistry with antibodies specific for epitopes associated with pectin, AGPs, and xyloglucan. Two antibodies, polygalacturonic acid (PGA)/rhamnogalacturonan I (RG1) and JIM 5, were used to locate epitopes associated with pectin. The polyclonal PGA/RG1 antibody was found to label throughout the cell walls of both hydrated and desiccated tissue (Fig. 8, A and B ), whereas the monoclonal JIM 5 antibody was found to specifically label the middle lamella region in both hydrated and desiccated cell walls (Fig. 8, C and D). The location of xyloglucan epitopes was investigated using an anti-XG polyclonal antibody. This antibody revealed material throughout hydrated and desiccated cell walls (Fig. 8, E and F) but failed to detect xyloglucan epitopes in the middle lamella zone and cell junctions (Fig. 8E). The monoclonal anti-arabinan LM 6 antibody directed against the (1  5)- -L-arabinan epitope (Willats et al., 1998) was shown to label material in the cell walls of both hydrated and desiccated leaf tissue in close proximity to the cytoplasm (Fig. 9, A and B
). Finally, the monoclonal anti-AGP JIM 13 antibody was shown to predominantly label the cell cytoplasm near the plasma membrane (Fig. 9, C and D) in both desiccated and hydrated leaf tissue. Our data showed that, whereas LM 6 labeled the cell wall adjacent to the plasma membrane (Fig. 9, A and B), JIM 13 predominantly labeled the cytoplasm and plasma membrane near the cell wall (Fig. 9, C and D).
Additional analysis of pectin epitopes was performed using the monoclonal JIM 7 antibody, which detects homogalacturonans possessing a relatively high level of methylesterification with flanking unesterifed GalUA residues (Willats et al., 2000 ; Clausen et al., 2003) as well as the monoclonal LM 7 antibody, which recognizes relatively unesterified homogalacturonans with flanking methyl-esterified GalUA residues (Clausen et al., 2003). These antibodies did not reveal any data (data not shown) additional to those found with the antibodies already used.
Electron microscopic analysis of desiccation-induced M. flabellifolius leaf cell wall folding revealed distinct folding in mesophyll and epidermal cells accompanied by cytoplasm shrinkage and possible crystallization presumably due to water loss. We believe that the absence of folding seen with gland, vascular, and sclerenchyma cells is due to these cells having thick, possibly reinforced, walls. In particular, the majority of the cells where wall folding occurs upon desiccation sit between the sclerenchymous ribs (Grundell, 1933 ), which form a fan-like structure. It is likely that these thick-walled cells and tissues provide structural support to the leaf during desiccation, thereby allowing leaf morphology to be rapidly regained upon rehydration.
Dicotyledonous plants generally contain cell walls with about one-third each of cellulose, hemicellulose, and pectin components (Brett and Waldron, 1996
The locations of the various epitopes were investigated by immunocytochemistry. No major deviations from the normal patterns of epitope found in other plants were observed in M. flabellifolius leaves and no major change in the location of epitopes between hydrated and desiccated material was noted. Our immunological data using the LM 6 antibody showed that (1
M. flabellifolius leaves were found to have an unusually large amount of Ara present, presumably in the form of arabinan polymers. Arabinose polymers have been shown to be highly mobile (Foster et al., 1996
Plant Material
Myrothamnus flabellifolius plants, collected from the Buffelskloof Nature Reserve, Mpumalanga Province, South Africa, were maintained in a glasshouse in the Botany Department, University of Cape Town. Desiccation of whole plants was performed by withholding water and allowing the plants to dry naturally under ambient environmental conditions. RWC measurements were performed as outlined previously (Sherwin and Farrant, 1996
Scanning electron microscopy was performed using a Leica Stereoscan 440 digital scanning electron microscope equipped with a Fisons LT7400 Cryo transfer system. Leaves from hydrated and desiccated plants were frozen using liquid nitrogen and viewed directly or after freeze fracturing. Transmission electron microscopy was performed using a LEO 912 transmission electron microscope equipped with a CCD camera. Leaf segments (1–2 mm2) were excised from the midblade of hydrated or desiccated leaves and fixed overnight at 4°C in 0.1 M phosphate buffer, pH 7.4, containing 2.5% glutaraldehyde supplemented with 0.5% caffeine. Fixed samples were dehydrated in ethanol and embedded in epoxy resin (Spurr, 1969
Cell walls were prepared from flash frozen and lyophilized hydrated (RWC approximately 86%) and desiccated (RWC approximately 9%) leaves from hydrated and desiccated plants. The lyophilate was ground to a fine powder using a pestle and mortar and suspended in boiling ethanol (1% w/v) for 15 min to deactivate any enzymes present. The powder was recovered by filtration and subjected to a series of extractions to remove lipids, polyphenols, and other low-Mr metabolites. Briefly, the residues were extracted for 12 h at room temperature twice with methanol-chloroform (1:1; v/v), twice with methanol-acetone (1:1; v/v), and finally with acetone-water (4:1; v/v). The residue was air dried at 80°C, suspended in 50 mM acetate, pH 5.4, and destarched at 80°C using a thermostable
A scheme outlining the fractionation procedure, based on that developed by Selvendran (1985
The monosaccharide composition of each cell wall fraction apart from the H2SO4 hydrolysate was analyzed by gas liquid chromatography (York et al., 1985
Xyloglucan polymer fragments were generated by digestion at 37°C for 24 h with 5 to 10 units of endo-
AGPs were extracted from lyophilized hydrated and desiccated leaf material as described previously (Schultz et al., 2000
One to 2 mm2 segments were excised from the midblade of hydrated and desiccated leaves and fixed overnight at 4°C in 0.1 M phosphate buffer, pH 7.4, containing 4% paraformaldehyde and 0.5% glutaraldehyde, supplemented with 0.5% caffeine. Fixed samples were dehydrated in ethanol, placed in beem capsules to exclude air, and infiltrated with LR White resin before being hardened by heating overnight at 60°C. Thin sections (90–100 nm) were prepared using a Reichert ultracut-S ultramicrotome and collected on 200-µm mesh formvar-backed nickel grids.
Antibodies used were selected to recognize specific epitopes. The polyclonal anti-PGA/RG1 antibody has been reported to recognize the nonesterified form of homogalacturonans in pectin (Moore et al., 1986
We thank Marc-Antoine Cannesan, Sophie Aboughe, Bimalendu Ray, and Christophe Rihouey for technical assistance provided to J.P.M. during his stay at the University of Rouen. We would also like to thank Miranda Waldron and Mohammed Jaffer (University of Cape Town electron microscope unit) for their excellent technical assistance, as well as Elizabeth Parker and John and Sandy Burrows from Buffelskloof Nature Reserve for the plant material. Received January 24, 2006; returned for revision March 14, 2006; accepted March 22, 2006.
1 This work was supported by the University of Cape Town, Deutscher Akademischer Austausch Dienst (Germany), University of Rouen, the National Research Foundation (financial assistance to J.P.M.), by the National Research Foundation and the University of Cape Town Research Council (research grants to J.M.F.), and by le Ministère de l'Enseignement Supérieur et de la Recherche et l'Université de Rouen (research grants to A.D.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: George G. Lindsey (lindsey{at}science.uct.ac.za). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.077701. * Corresponding author; e-mail lindsey{at}science.uct.ac.za; fax 27–21–689–7573.
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ABSTRACT
RESULTS
-(1,4) glucanase (EC 3.2.1.4; Megazyme International) in 1 mL of 50 mM sodium acetate, pH 5.4. These fragments were separated using a DIONEX HPLC and the masses determined by MALDI-TOF mass spectrometry using a Micromass mass spectrometer with dihydroxybenzoic acid as the matrix. Standards were prepared by digestion of tamarind (Tamarindus indica) seed (York et al., 1993