Response of the Leaf Cell Wall to Desiccation in the Resurrection Plant Myrothamnus ... · Response of the Leaf Cell Wall to Desiccation in the Resurrection Plant Myrothamnus ﬂabellifolius1

Response of the Leaf Cell Wall to Desiccation in theResurrection Plant Myrothamnus flabellifolius1

John P. Moore, Eric Nguema-Ona, Laurence Chevalier, George G. Lindsey*, Wolf F. Brandt,Patrice Lerouge, Jill M. Farrant, and Azeddine Driouich

Department 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, Unite Mixte de Recherche6037, Institut Federatif de Recherche Multidisciplinaire sur les Peptides 23, Centre Commun de MicroscopieElectronique, Universite 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 themajority of mesophyll and epidermal cells. Thick-walled vascular tissue and sclerenchymous ribs did not fold and supportedthe surrounding tissue, thereby limiting the extent of leaf shrinkage and allowing leaf morphology to be rapidly regained uponrehydration. Isolated cell walls from hydrated and desiccated M. flabellifolius leaves were fractionated into their constituentpolymers and the resulting fractions were analyzed for monosaccharide content. Significant differences between hydrated anddesiccated states were observed in the water-soluble buffer extract, pectin fractions, and the arabinogalactan protein-richextract. A marked increase in galacturonic acid was found in the alkali-insoluble pectic fraction. Xyloglucan structure wasanalyzed and shown to be of the standard dicotyledonous pattern. Immunocytochemical analysis determined the cellularlocation of the various epitopes associated with cell wall components, including pectin, xyloglucan, and arabinogalactan pro-teins, in hydrated and desiccated leaf tissue. The most striking observation was a constitutively present high concentration ofarabinose, which was associated with pectin, presumably in the form of arabinan polymers. We propose that the arabinan-richleaf cell wall of M. flabellifolius possesses the necessary structural properties to be able to undergo repeated periods ofdesiccation and rehydration.

Desiccation tolerance of vegetative plant tissue is aphenomenon found throughout the plant kingdom(Bewley and Krochko, 1982). It is more common amonglower plants, such as mosses and ferns, whereas it israrely observed in angiosperm families (Alpert andOliver, 2002). Plants that display desiccation toleranceare commonly called resurrection plants (Oliver, 1996),so named because of their ability to lose most of theircellular water (down to ,5% relative water content[RWC]), cease metabolic function, and exist in a qui-escent, desiccated state for extended periods and thenrehydrate their vegetative tissue and resume metabo-lism (Gaff, 1977). The largest resurrection plant isMyrothamnus 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 andMyrothamnus moschatus (Glen et al., 1999), divergedfrom the Gunnera, one of the oldest surviving angio-sperm genera, approximately 1.2 3 108 years ago andis therefore likely to have been one of the earliest an-giosperm families to have acquired desiccation toler-ance (Wanntorp et al., 2001). Mechanisms proposed toexplain the ability of resurrection plants to survivedesiccation include Suc and trehalose accumulation(Bianchi et al., 1993; Drennan et al., 1993), accumula-tion of stress proteins (Goyal et al., 2005;Mtwisha et al.,2006), as well as the presence of polyphenols, in par-ticular galloylquinic acids, which have been shown toact as antioxidants and membrane protectants (Mooreet al., 2005). These mechanisms have evolved to coun-teract stresses imposed during desiccation and subse-quent rehydration (Alpert and Oliver, 2002). The mostvisible result of desiccation is a considerable reductionin tissue and cell volume that occurs due to water loss(Farrant, 2000). Thus, the cell volume of Craterostigmawilmsii has been reported to be on the order of 78% lessupon desiccation (Farrant, 2000) and is accompaniedby a concomitant withdrawal of the plasmamembranefrom the cell wall as well as extensive cell wall folding(Vicre et al., 2004b) believed to result in mechanicalstress (Farrant, 2000; Vicre et al., 2004a). Mechanicalstress is defined as the tension that develops between

1 This work was supported by the University of Cape Town,Deutscher Akademischer Austausch Dienst (Germany), Universityof Rouen, the National Research Foundation (financial assistance toJ.P.M.), by the National Research Foundation and the University ofCape Town Research Council (research grants to J.M.F.), and by leMinistere de l’Enseignement Superieur et de la Recherche et l’Uni-versite de Rouen (research grants to A.D.).

* Corresponding author; e-mail [email protected]; fax27–21–689–7573.

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:George G. Lindsey ([email protected]).

Article, publication date, and citation information can be found atwww.plantphysiol.org/cgi/doi/10.1104/pp.106.077701.

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and within the plasma membrane and the cell walldue to a loss of turgor pressure, resulting in plasmol-ysis (Walters et al., 2002). The damage resulting frommechanical stress is dependent on factors such as thestrength of plasmalemma-cell wall contacts, degree ofplasma membrane disruption, as well as elasticity ofthe cell wall (Walters et al., 2002). It has been proposed(Iljin, 1957) that plants must overcome such mechan-ical stress to acquire desiccation tolerance. Strategiesemployed by desiccation-tolerant organisms to coun-teract mechanical stress include storage within vacu-oles of proteins, lipids, and carbohydrates to replacelost water and cell wall folding to prevent possiblerupture of the plasma membrane (Vicre et al., 2004a).Desiccation-induced wall folding has been proposed(Webb and Arnott, 1982) to be essential for structuralpreservation of tissue and the extent and manner ofsuch folding is species specific and dependent uponthe chemical composition and molecular architectureof the cell wall.

Plant cell walls are dynamic entities that governthe morphology, growth, and development of plants(Albersheim et al., 1994). They also mediate the inter-action between the cell and its environment (Penell,1998), which includes environmental stresses such aswounding (Cardemil and Riquelme, 1991), osmoticstress (Wakabayashi et al., 1997), cold acclimation(Weiser et al., 1990), drought tolerance (Zwiazek,1991), and pathogen invasion (Boudart et al., 1998).The molecular architecture of plant cell walls is im-perfectly understood (Reiter, 1998), although recentresearch with Arabidopsis (Arabidopsis thaliana) mu-tants and transgenic plants has revealed new insightsinto the complex nature of the plant cell wall (Reiteret al., 1997; Shevell et al., 2000; His et al., 2001, Lerouxelet al., 2002; Reiter, 2002; Mouille et al., 2003). Resur-rection plants provide unique model systems to in-vestigate the relationship between desiccation stressand the cell wall (Vicre et al., 2004a). In addition,resurrection plants display tolerance to other abioticstresses, such as hypersalinity and high temperature(Gaff andWood, 1988; Gaff, 1989; Hartung et al., 1998).This is not surprising because acquiring tolerance to aspecific stress often results in some tolerance of relatedstresses (Mowla et al., 2002) due to cross talk in stressresponse pathways (Bartels and Salamini, 2001). Thisreport has investigated the composition andmoleculararchitecture of the cell wall of M. flabellifolius leavesbefore and after desiccation using biochemical andimmunocytochemical approaches. The results of thisstudy revealed folded cell walls in the majority ofdesiccated leaf cells of leaf tissue, implicating wallfolding as the major mechanism by which M. flabelli-folius minimizes mechanical damage. The biochemicaland immunocytochemical analysis showed that thecell wall of M. flabellifolius underwent desiccation-induced modifications and generally conformed to thestandard pattern found in dicotyledonous plant cellwalls, except that the wall contained an unusualabundance of Ara most likely in the form of pectin-

associated arabinan polymers. We propose that thisarabinan-rich wall is well adapted to be able to with-stand cycles of desiccation and hydration.

RESULTS

The pronounced morphological changes observedon desiccation of M. flabellifolius are similar to thoseobserved in other resurrection plants undergoing thesame process (Alpert and Oliver, 2002; Vicre et al.,2004a). Dehydrating M. flabellifolius plants undergocurling of both their stem segments as well as theirleaves (Fig. 1). In particular, the leaves fold upward toa position where the adaxial surfaces are appressedagainst the stem (Fig. 1) and the sclerenchymous leafribs (Grundell, 1933) move closer to one another, there-by closing the leaf face, which has been proposed tominimize the exposure of the desiccated leaf to light(Farrant et al., 2003). Electron microscopy of hydratedand desiccated leaves revealed further morphologicalchanges that occurred upon desiccation (Fig. 2, A–H).Cryoscanning electron microscopy of the epidermalsurface of hydrated leaves revealed a distinctive net-work pattern of stomata and gland cells interdispersedbetween regular epidermal cells (Fig. 2A). Upon des-iccation, these epidermal cells underwent massive cellwall folding and shrinkage around the seemingly lessflexible stomata and gland cells (Fig. 2B). The stomatain particular were clearly visible in the desiccatedstate, displaying little of the extreme morphologicalchanges present in desiccated epidermal cells (Fig. 2B).Freeze-fracture cross sections through cryofixed leavesshowed that considerable changes in tissue and cellu-lar structure occurred associated with the desiccationprocess (Fig. 2, C–F). Electron microscopy of a crosssection through a frozen hydrated leaf (Fig. 2, C and E)revealed clear anatomical features, such as the epider-mis, spongy mesophyll layer, palisade mesophylllayer, as well as vascular cells. In contrast, electronmicroscopy of a cross section through an identically

Figure 1. Top view of the resurrection plant M. flabellifolius in thedesiccated (A) and the hydrated (B) state.

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treated desiccated leaf showed no discernible anatom-ical organization (Fig. 2, D and F). It would appear thatcell wall folding had occurred during desiccation,resulting in a compacted, wrinkled appearance of theleaf cell layers. Only sclerenchymous and vascularcells resistant to folding, as well as epidermal cells,were clearly identifiable (Fig. 2, D and F). Highermagnification examination of hydrated tissue (Fig. 2E)revealed turgid endodermal cells and small (approx-imately 30-mm diameter) spongy mesophyll cells withmultiple plasmodesmata connections to adjacent cells.Several intercellular airspaces and calcium oxalatedruse crystals (Grundell, 1933) were also clearly vis-ible. Desiccated leaf tissue at the same magnification(Fig. 2F) showed none of the features evident in hy-drated tissue. Only folded cell wall fragments, mostlikely the result of mechanical fracturing during sam-ple preparation, a greater number of intercellular air-spaces, and amorphous deposits between cell wall

layers, were observed. These amorphous deposits werepossibly the result of desiccation of salts and cytosolicconstituents.

Examination of chemically fixed hydrated and des-iccated leaf tissue by transmission electron microscopyprovided further evidence for the changes observedusing 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, starchgranules, and large central vacuoles. The cytoplasm ofthese cells occurred on the periphery of the cellsadjacent to the cell wall. In contrast, transverse sec-tions of desiccated leaves (Fig. 2H) showed cells withfolded cell walls and a compact dense cytoplasmseparated from the cell wall in a manner that resem-bled that observed in plasmolyzed plant cells, althoughthe plasmalemma remained intact. The plasmolyzed

Figure 2. Scanning (A–F) and transmission (G and H)electron micrographs of hydrated (A, C, E, and G) anddesiccated (B, D, F, and H) M. flabellifolius leaves.Surface view of the epidermal surface of hydratedand desiccated leaves (A and B); transverse sectionsof hydrated and desiccated leaves (C–H). Note thatmicrographs E and F are at higher magnification thanthose shown in C and D. Transmission electron micro-graphs: Transverse sections of hydrated (G) and des-iccated (H) leaves. c, Calcium oxalate crystal; chl,chloroplast; cp, cytoplasm; cw, cell wall; en, endo-dermis; g, gland; is, intercellular space; n, nucleus;pd, plasmodesmata; pm, palisade mesophyll; sg,starch granule; st, stomata; sm, spongy mesophyll;v, vacuole; vb, vascular bundle; /, wall folding.Scale bars: A and B 5 70 mm; C 5 100 mm; D 5

80 mm; E and F 5 40 mm; G and H 5 3 mm.

Cell Wall of Myrothamnus flabellifolius

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appearance of desiccated cells might have beenbrought about by use of an aqueous aldehyde-basedfixative (Hayat, 1981). This would preferentially fixcytoplasmic proteins rather than the cell wall carbo-hydrates because the former have a higher number ofamino groups (Hayat, 1981). Because desiccated cellwalls would presumably have swelled during thefixation process, our observations of cell wall foldingin fixed desiccated cells provide strong evidence thatcell wall folding occurs in vivo, and this is probablythe main mechanism of mechanical stabilization of themesophyll cells of this species.

Because the cell wall of M. flabellifolius might beunique on account of its taxonomic position (Qiu et al.,1998) being basally situated in angiosperm evolution,we next investigated the composition of the cell wallof both hydrated and desiccated leaves. Hydratedor desiccated leaf material was serially extracted witha variety of organic solvents (see ‘‘Materials andMethods’’) before being air dried; this material repre-sented total cell walls of these leaves. No differencewas detected in the recovery of cell wall material fromhydrated leaves (42% 6 5%) and desiccated leaves(40% 6 5%). The total sugar composition of these cellwalls was determined (Fig. 3) after trifluoroacetic acid(TFA) hydrolysis (York et al., 1985). This analysis,which excluded the TFA-resistant a-crystalline cellu-lose, revealed Ara, Xyl, and galacturonic acid (GalUA)as the predominant monosaccharides, representingapproximately 75% of the total hydrolyzable sugarpresent in both hydrated and desiccated leaves (Fig. 3).The other monosaccharides present, in decreasingorder of concentration, in both hydrated and desic-cated leaves were found to be Gal, Glc, Rha, Man,GlcUA, and Fuc. Of all these monosaccharides, onlyXyl and GalUA displayed any concentration differ-ences between hydrated and desiccated leaves with an

elevated level of Xyl and a decreased level of GalUApresent in desiccated leaves.

We next fractionated the total cell walls preparedfrom hydrated and desiccated M. flabellifolius leaves(Table I; Fig. 4) by serial extraction, initially withphosphate buffer at 80�C, then with CDTA, and finallywith increasing concentrations of KOH. These latterextractions were all performed at 20�C. Each fractionextracted (A–I; Table I) was gravimetrically analyzedbefore being hydrolyzed with TFA; insoluble materialremaining after TFA hydrolysis of fraction I was hy-drolyzed with H2SO4 (fraction J). These hydrolysateswere then analyzed for the presence of individualmonosaccharides (Fig. 5). Gravimetric analysis of ly-ophilized fractions recovered from serial fractionation(Table I) yielded three significant differences in frac-tions B, D, and E between hydrated and desiccatedleaves. CDTA extraction (fraction B) extracted morematerial from hydrated (10.9% 6 0.4%) than fromdesiccated (8.0% 6 0.5%) samples; more 0.05 M KOHinsoluble material (fraction D) was present in desic-cated (12.3%6 1.5%) comparedwith hydrated (9.7%60.4%) samples and 1 M KOH (fraction E) extractedalmost double thematerial fromhydrated (8.2%6 1.6%)than from desiccated (4.1% 6 0.6%) samples.

The monosaccharide composition of each fractionobtained after cell wall fractionation was determined(Fig. 5), allowing the ability to infer the main polymerspresent. Fraction A contained predominantly Ara,GalUA, Man, and Gal, consistent with mannoproteins,arabinogalactan proteins (AGPs), soluble pectin, andother glycoproteins being extracted with phosphatebuffer at 80�C. There were significant differences in thelevels of GalUA, Gal, and Ara between hydrated anddesiccated leaves, with hydrated leaves found to con-tain increased levels of GalUA and decreased levels ofGal and Ara. Fraction B contained predominantly Araand GalUA, consistent with CDTA-mediated solubili-zation of pectin. The presence of Ara suggested theassociation of neutral arabinan chains together withthe pectin, in agreement with previous data presented(Fig. 3), which revealed high Ara and GalUA contentin the cell wall. Furthermore, less Ara was present inhydrated compared with desiccated leaves. Fraction Ccontained Ara, GalUA, Glc, Rha, and Xyl as the majormonosaccharides present. Of these monosaccharides,relatively increased amounts of Xyl and Glc and adecreased amount of GalUAwere found to be presentin hydrated leaves. Fraction D contained Ara, GalUA,and Rha as the most abundant monosaccharides pre-sent, with increased levels of Ara and Rha and adecreased level of GalUA found in hydrated leaves.The high amounts of Ara and GalUA present in thesefractions (C and D) indicated that additional pecticmaterial is present in desiccated leaves associated witharabinans tightly bound to the cell wall and onlyreleased by strong alkali extraction. Rhamnogalactur-onan polymers were also inferred to be present dueto the presence of Rha in both extracts. Fraction Econtained primarily Xyl, with lesser amounts of Ara

Figure 3. Monosaccharide composition of total cell walls of hydrated(white bars) and desiccated (gray bars) leaves. Monosaccharides ana-lyzed were Ara, Rha, Fuc, Xyl, GlcUA, GalUA, Man, Gal, and Glc.Error bars represent SDs of the mean from two independent experimentswith at least three replicates per experiment.

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and Glc, together with small amounts of the othermonosaccharides, consistent with the presence ofarabinoxylans in this fraction. Hydrated leaves werefound to contain increased Xyl and Glc and decreasedAra concentrations. Fraction F contained almost ex-clusively Xyl, with no significant difference in concen-tration between the hydrated and desiccated states.The high concentration of Xyl in both the soluble andinsoluble 1 M KOH extracts (fractions E and F) sug-gested the presence of the more soluble arabinoxylansin fraction E and the insoluble crystalline xylan poly-mers in fraction F. Continued serial extraction with 4 M

KOH and subsequent TFA hydrolysis failed to revealany significant differences between hydrated and des-iccated leaves. Material extracted with 4 M KOH (frac-tion G) contained mainly Xyl and Glc, together withlesser amounts of Ara, Man, and Gal, consistent withthe presence of xyloglucan polymers. Insoluble mate-rial after extraction with 4 M KOH (fraction H) con-tained chiefly Xyl, suggesting the presence of residualinsoluble crystalline xylans. The alkali-insoluble resi-due remaining after serial extraction was finally sub-jected to successive acid hydrolysis using first TFA(fraction I) and then H2SO4 (fraction J). Fraction I wasfound to contain high amounts of Ara, together withlesser amounts of GalUA and Xyl, suggesting the pre-sence of residual tightly bound pectic arabinans andxylans. Fraction J contained mostly Glc, most likelyderived from crystalline cellulose. Mannans have beenreported to be associated with cellulose (Fry, 1988); thepresence of Man in this fraction suggested such anassociation in M. flabellifolius leaves.To characterize the xyloglucan component of M.

flabellifolius leaf hemicellulose, the major load-bearingpolymers of the plant cell wall, isolated cell walls, weresubjected 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 degradationresulted 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/z1,084 ion was assigned to XXXG (nomenclature ac-cording to Fry et al. [1993]), with the m/z 1,288, 1,435,

Figure 4. Flow diagram of the cell wall fractionation procedure. Allextractions were for 2 h at room temperature, except the hot bufferextraction, which was at 80�C. Alkaline extractions were supplementedwith 1% NaBH4 and performed under a nitrogen atmosphere.

Table I. Gravimetric analysis of material isolated as a result of the fractionation procedure (Fig. 4)from hydrated and desiccated leaf cell walls

KOH A and KOH B represent soluble and insoluble components, respectively. Fractions A to I werehydrolyzed with TFA; insoluble material remaining after TFA hydrolysis of fraction I was hydrolyzed withH2SO4 (fraction J). The column Component Extracted describes the major polymers proposed to be presentin each fraction. The data represent g/100 g dry cell wall material of duplicate samples from twoindependent experiments with two replicates per experiment. Error values represent SDs of the mean.

Fraction Extraction Method Component Extracted Hydrated Desiccated

A Hot buffer Proteins and pectin 6.9 6 2.1 7.6 6 2.8B CDTA Pectin and neutral polymers 10.9 6 0.4 8.0 6 0.5C 0.05 M KOH A Pectin and neutral polymers 3.4 6 1.7 6.0 6 2.0D 0.05 M KOH B Pectin and neutral polymers 9.7 6 0.4 12.3 6 1.5E 1 M KOH A Xylans and neutral polymers 8.2 6 1.6 4.1 6 0.6F 1 M KOH B Xylans 5.1 6 0.3 4.9 6 0.2G 4 M KOH A Xyloglucan 6.3 6 0.3 7.2 6 1.1H 4 M KOH B Xylans and neutral polymers 5.4 6 0.7 4.4 6 1.1I and J Insoluble residue

(including TFA extract)Cellulose and neutral polymers 44.2 6 5.6 45.6 6 7.4





Figure 5. Monosaccharide composition (see Fig. 3 for abbreviations used) of cell wall fractions prepared as per Figure 4 fromhydrated (white bars) and desiccated (gray bars) leaves. A, Hot buffer. B, CDTA. C, 50 mM KOH A. D, 50 mM KOH B. E, 1 M KOHA. F, 1 M KOH B. G, 4 M KOH A. H, 4 M KOH B. I, TFA hydrolysates. J, H2SO4 hydrolysate. A and B suffixes refer to soluble andinsoluble material, respectively. Error bars represent SDs of the mean from two independent experiments with at least threereplicates per experiment.




and 1,639 ions assigned to monoacetylated XXLG,monoacetylated XXFG, and diacetylated XLFG, re-spectively. These were confirmed by MALDI-TOFmass spectrometry after deacetylation using sodiumhydroxide that resulted in ions of m/z 1,247, 1,393, and1,555, respectively (data not shown). Traces of theseunacetylated parent oligosaccharide ions are presentin the original mass spectrum (Fig. 6). Although theposition of acetylation was not determined, it is likelyto occur on Gal residues by analogy with previousstudies on xyloglucan acetylation (Fry, 1988). Nomajordifferences in ion composition or intensity were ob-served between enzymatic extracts sourced from hy-drated or desiccated cell walls (Fig. 6).

AGPs have been reported to be associated with thecell wall and plasma membrane of plant cells (Knox,1997), where it has been proposed that they function ina structural capacity likely to facilitate important pro-cesses in plant growth and development (Showalter,2001). A study of the monosaccharide content of AGPsisolated using the Yariv reagent (Schultz et al., 2000)from hydrated and desiccated M. flabellifolius leaftissue was performed. This showed an abundance ofAra, Gal, GalUA, and Man from both hydrated anddesiccated leaves with elevated levels of GalUA andGal observed in hydrated samples (Fig. 7). In addi-tion, quantification of Yariv-precipitable AGPs usingrocket electrophoresis did not reveal any difference

Figure 6. Mass spectral analysis of xyloglucan frag-ments of hydrated (A) and desiccated (B) leaves.XXXG, XXLG, XXFG, and XLFG denote hepta-saccharide, octa-saccharide, nona-saccharide, anddeca-saccharide fragments, respectively, accordingto the standard xyloglucan nomenclature (Fry et al.,1993). The ordinate denotes the relative percentageion abundance.





between hydrated and desiccated samples (data notshown).

The distribution of various carbohydrate epitopespresent in or near the cell walls of hydrated and des-iccated M. flabellifolius leaves was next investigatedusing immunocytochemistry with antibodies specificfor epitopes associated with pectin, AGPs, and xylo-glucan. Two antibodies, polygalacturonic acid (PGA)/rhamnogalacturonan I (RG1) and JIM 5, were used tolocate epitopes associated with pectin. The polyclonalPGA/RG1 antibody was found to label throughout thecell walls of both hydrated anddesiccated tissue (Fig. 8,A and B), whereas the monoclonal JIM 5 antibody wasfound to specifically label the middle lamella region inboth hydrated and desiccated cell walls (Fig. 8, C andD). The location of xyloglucan epitopes was investi-gated using an anti-XG polyclonal antibody. This anti-body revealed material throughout hydrated anddesiccated cell walls (Fig. 8, E and F) but failed to detectxyloglucan epitopes in themiddle lamella zone and celljunctions (Fig. 8E). Themonoclonal anti-arabinan LM 6antibody directed against the (1/5)-a-L-arabinan ep-itope (Willats et al., 1998)was shown to labelmaterial inthe cell walls of both hydrated and desiccated leaftissue in close proximity to the cytoplasm (Fig. 9, A andB). Finally, the monoclonal anti-AGP JIM 13 antibodywas shown to predominantly label the cell cytoplasmnear the plasma membrane (Fig. 9, C and D) in bothdesiccated and hydrated leaf tissue. Our data showedthat, whereas LM 6 labeled the cell wall adjacent to theplasma membrane (Fig. 9, A and B), JIM 13 predomi-nantly labeled the cytoplasm and plasma membranenear the cell wall (Fig. 9, C and D).

Additional analysis of pectin epitopes was per-formed using the monoclonal JIM 7 antibody, whichdetects homogalacturonans possessing a relativelyhigh level of methylesterification with flanking unes-terifed GalUA residues (Willats et al., 2000; Clausen

et al., 2003) as well as the monoclonal LM 7 antibody,which recognizes relatively unesterified homogalac-turonans with flanking methyl-esterified GalUA resi-dues (Clausen et al., 2003). These antibodies did notreveal any data (data not shown) additional to thosefound with the antibodies already used.

DISCUSSION

Electron microscopic analysis of desiccation-induced M. flabellifolius leaf cell wall folding revealeddistinct folding in mesophyll and epidermal cells ac-companied by cytoplasm shrinkage and possible crys-tallization presumably due to water loss. We believethat the absence of folding seen with gland, vascular,and sclerenchyma cells is due to these cells havingthick, possibly reinforced, walls. In particular, themajority of the cells where wall folding occurs upondesiccation sit between the sclerenchymous ribs(Grundell, 1933), which form a fan-like structure. Itis likely that these thick-walled cells and tissues pro-vide structural support to the leaf during desiccation,thereby allowing leaf morphology to be rapidly re-gained upon rehydration.

Dicotyledonous plants generally contain cell wallswith about one-third each of cellulose, hemicellulose,and pectin components (Brett and Waldron, 1996). In

Figure 7. Monosaccharide composition (see Fig. 3 for abbreviationsused) of purified Yariv-precipitable AGPs isolated from hydrated (whitebars) and desiccated (gray bars) leaves. Error bars represent SDs of themean from two independent experiments with at least three replicatesper experiment.

Figure 8. Immunogold labeling of pectin and xyloglucan epitopespresent in hydrated (A, C, and E) and desiccated (B, D, and F) leaf cellwalls. The epitopes detected were homogalacturonan using the PGA/RG1 antibody (A and B) and the JIM 5 antibody (C and D), andxyloglucan using the anti-XG antibody (E and F). cp, Cytoplasm; cw,cell wall; is, intercellular space; ml, middle lamella. Scale bars: A andB 5 400 nm; C and D 5 300 nm; E 5 300 nm; F 5 150 nm.

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contrast, M. flabellifolius cell walls were found to con-tain approximately 45% cellulose, 25% hemicellulose,and 30% pectin. We propose that the vascular tissueand ribs contributed to the large cellulose fraction ob-served. Analysis of the alkali-insoluble pectin showeda marked increase in GalUA content upon desiccation.This is interesting because it correlates with an immu-nocytochemical study of desiccation-induced changesin the cell wall of the resurrection plant C. wilmsii,which showed that pectin epitopes increased upondesiccation (Vicre et al., 1999). A further biochemicalstudy of the cell wall of C. wilmsii revealed no overallchange in GalUA content between states, but did re-veal a change in solubility of pectin polymers as de-termined by differential fractionation from hydratedand desiccated cell walls (Vicre et al., 2004b). Inrelation to M. flabellifolius, the additional pectin mate-rial recovered might be due to an increased solubili-zation of pectins from cell walls of desiccated plants.As proposed for C. wilmsii (Vicre et al., 2004b), our datasuggest that there is a change in the physical proper-ties of the cell wall of M. flabellifolius following desic-cation. In addition, an unusual abundance of Ara,possibly in the form of arabinans, was observed asso-ciated with pectic polymers during sequential extrac-tions of cell walls isolated from desiccated plants.Precipitation of AGPs using the Yariv reagent failed toreveal any major differences in quantity and compo-sition between hydrated and desiccated leaf tissue.These data do not rule out changes in quantity andcomposition of AGPs that were not precipitated usingthe Yariv reagent.The locations of the various epitopes were investi-

gated by immunocytochemistry. No major deviationsfrom the normal patterns of epitope found in other

plants were observed in M. flabellifolius leaves and nomajor change in the location of epitopes between hy-drated and desiccated material was noted. Our im-munological data using the LM 6 antibody showedthat (1/5)-a-L-arabinan epitopes are present in theprimary cell walls of the leaf tissue. In contrast, thedata using the JIM 13 antibody showed that the AGPepitopes recognized by this antibody are present in thecytoplasm adjacent to the plasma membrane. The(1/5)-a-L-arabinan epitopes were detected adjacentto the cytoplasm and are most probably associatedwith either RG1 or with AGPs distinct from thoserecognized by JIM 13. The pattern of distribution ofLM 6 epitopes has been observed previously with thisantibody in other plant species (Willats et al., 1998;Orfila et al., 2001). Vascular and sclerenchyma tissuenormally contain thick secondary cell walls composedof relatively high concentrations of cellulose and lignincompared with pectin. Since no (1/5)-a-L-arabinanepitopes were detected in vascular and sclerenchymatissue using the LM 6 antibody, it is unlikely that thesetissues contain high quantities of polymers containingAra in this linkage, although other forms of Ara-containing polymers may be present. The hemicellu-lose component of the cell wall of M. flabellifolius wasfound to contain a large amount of xylan and lesseramounts of xyloglucan polymers, which showed ahigh degree of acetylation as has been shown in otherdicotyledonous plants. Neither the composition northe structure of the xyloglucan present changed upondesiccation. The presence of xyloglucan anti-XG epi-topes reinforces the biochemical analysis previouslydiscussed.

M. flabellifolius leaves were found to have an unusu-ally large amount of Ara present, presumably in theform of arabinan polymers. Arabinose polymers havebeen shown to be highly mobile (Foster et al., 1996;Renard and Jarvis, 1999) and to have a high water-absorbing capacity (Goldberg et al., 1989; Belton, 1997),properties ideally suited to cell wall rehydration. Ourdata showed no overall difference in the Ara levelsbetween hydrated and desiccated leaves, suggestingthat Ara is constitutively synthesized and that the M.flabellifolius cell wall is continuously prepared for lossof water. This is interesting because study of the cellwall response to hypersalinity, 0.4 M NaCl for 2 weeks,in the drought- and salt-resistant Mesembryanthemumcrystallinum, showed that some wall remodeling oc-curred, but the major observation was a high concen-tration of Ara, around 60% of the noncellulosic sugars,consistently present in the cell wall of both treated andnontreated plants (Galkina et al., 2001). It has recentlybeen shown that arabinan polymers act as plasticizers,increasing cell wall flexibility and diminishing stronginteractions between homogalacturonan chains inpectin (Jones et al., 2003). Recently, potato (Solanumtuberosum) tuber tissue genetically engineered to bedeficient in arabinan and galactan side chains in cellwall pectin was shown to be significantly more brittleto imposed stress, highlighting the critical role that

Figure 9. Immunogold labeling of arabinan and AGP epitopes presentin hydrated (A and C) and desiccated (B and D) leaf cell walls. Theepitopes detected were linear (1/5)-a-L-arabinans using the LM 6antibody (A and B) and AGPs using the JIM 13 antibody (C and D). cp,Cytoplasm; cw, cell wall; is, intercellular space; ml, middle lamella;pm, plasma membrane; /, plasma membrane location. Scale bars:A 5 300 nm; B 5 150 nm; C 5 200 nm; D 5 200 nm.





these side chains play in controlling the biophysicalproperties of the cell wall (Ulvskov et al., 2005). Inaddition, arabinogalactan polymers in the form ofAGPs are believed to function in cell lubrication (Yateset al., 1996) and have been proposed to act as cell wallplasticizers (Lamport, 2001; Lee et al., 2005). A similarrole has been proposed for the stress response proteinHsp 12 in yeast (Saccharomyces cerevisiae; Motshweneet al., 2004; Karreman et al., 2005). Another possiblerole for a protein in wall flexibility was recently foundin the resurrection plant Craterostigma plantagineum,where a-expansin mRNA was demonstrated to beup-regulated in response to desiccation (Jones andMcQueen-Mason, 2004). These findings might there-fore indicate a role for AGPs in enhancing call wallflexibility. We believe that the high concentration ofAra polymers associated with the pectin matrix in theM. flabellifolius cell wall is likely to be one of the majorcomponents that allow the wall to desiccate and foldwithout any apparent damage.

MATERIALS AND METHODS

Plant Material

Myrothamnus flabellifolius plants, collected from the Buffelskloof Nature

Reserve, Mpumalanga Province, South Africa, were maintained in a glass-

house 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 and Transmission Electron Microscopy

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. Trans-

mission 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% glutar-

aldehyde supplemented with 0.5% caffeine. Fixed samples were dehydrated

in ethanol and embedded in epoxy resin (Spurr, 1969). Thin sections (90–100 nm)

were cut using a Reichert ultracut-S ultramicrotome and collected on 200-mm

mesh copper grids. Sections were stained with uranyl acetate and lead citrate

as described previously (Reynolds, 1963).

Isolation and Fractionation of Cell Wall Material

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-amylase and amyloglucosidase (EC

3.2.1.1; Megazyme International). Approximately 35% of the lyophilized leaf

weight was recovered.

A scheme outlining the fractionation procedure, based on that developed

by Selvendran (1985; Selvendran and O’Neill, 1987) has been provided (Fig. 4).

Briefly, cell wall material was subjected to a sequential extraction regime using

0.1 M phosphate, pH 6, at 80�C, 50 mM CDTA, pH 6.5, 50 mM KOH, 1 M KOH,

and 4 M KOH. All extractions were for 2 h at room temperature, except where

stated. CDTA extracts were dialyzed against 1 M NaCl to exchange the

CDTA anion for the chloride anion. Alkaline extractions supplemented with

1% NaBH4 were performed in a nitrogen atmosphere after which the pH

was adjusted to pH 4.5 with glacial acetic acid. All extracts were dialyzed

against distilled water, concentrated by rotary evaporation before being

lyophilized.

Composition Analysis of Cell Wall Fractions

The monosaccharide composition of each cell wall fraction apart from the

H2SO4 hydrolysate was analyzed by gas liquid chromatography (York et al.,

1985) usingmannitol as the internal standard. Inositol was used as the internal

standard for the H2SO4 hydrolysate. Briefly, each fraction (5–10 mg) was

hydrolyzed (2 M TFA, 110�C, 2 h) and the liberated monosaccharides

converted to the methoxy sugars by incubation at 80�C for 24 h in 1 M meth-

anolic HCl. After sialylation at 80�C for 30 min, samples were dried, dissolved

in cyclohexane/pyridine (50:1; v/v), and analyzed using a GC 3800 Varian gas

chromatography system equipped with a DB1 capillary column and a flame

ionization detector. A temperature program optimized for separation of the

most common cell wall monosaccharides, specifically, Ara, Fuc, Gal, GalUA,

Glc, GlcUA, Man, Rha, Xyl, as well as the internal standards inositol and

mannitol, was used. Chromatographic data were analyzed and integrated

using Varian GC Star Workstation software with the quantity of each mon-

osaccharide corrected according to its response factor.

Analysis of Cell Wall Xyloglucan

Xyloglucan polymer fragments were generated by digestion at 37�C for

24 h with 5 to 10 units of endo-b-(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 dihydroxy-

benzoic acid as the matrix. Standards were prepared by digestion of tamarind

(Tamarindus indica) seed (York et al., 1993) and Argania spinosa (Ray et al., 2004)

xyloglucan.

Extraction and Analysis of AGPs

AGPs were extracted from lyophilized hydrated and desiccated leaf

material as described previously (Schultz et al., 2000) before Yariv-precipitable

AGPs were selectively precipitated (Ding and Zhu, 1997; Schultz et al., 2000)

using the b-D-glucosyl Yariv reagent (Yariv et al., 1962; Biosupplies). Purified

Yariv-precipitable AGPs were hydrolyzed with TFA and analyzed for mon-

osaccharide composition as described previously for cell wall material.

Immunocytochemistry

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-mm

mesh formvar-backed nickel grids.

Antibodies used were selected to recognize specific epitopes. The poly-

clonal anti-PGA/RG1 antibody has been reported to recognize the nonester-

ified form of homogalacturonans in pectin (Moore et al., 1986; Lynch and

Staehelin, 1992), the monoclonal antibody JIM 5 has been reported to recog-

nize homogalacturonan regions displaying a relatively low degree of methyl

esterification with flanking residues of methyl-esterifed GalUA (VandenBosch

et al., 1989; Knox et al., 1990; Willats et al., 2000; Clausen et al., 2003), the

polyclonal anti-XG antibody has been reported to recognize b(1/4)-glucans

in the xyloglucan backbone (Moore et al., 1986), and the monoclonal antibody

LM 6 has been reported to specifically recognize the linear chains of (1/5)-a-

L-arabinan, which are known to be associated with pectin and AGPs (Knox,

1997; Willats et al., 1998). The monoclonal antibody JIM 13 has been shown to

recognize polysaccharide epitopes of AGPs (Knox et al., 1991; Yates et al.,

1996; Knox, 1997). Immunocytochemistry was performed essentially as de-

scribed previously (Vicre et al., 1999).

Moore et al.




ACKNOWLEDGMENTS

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; revised March 14, 2006; accepted March 22, 2006;

published April 7, 2006.

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Response of the Leaf Cell Wall to Desiccation in the Resurrection Plant Myrothamnus ... · Response of the Leaf Cell Wall to Desiccation in the Resurrection Plant Myrothamnus ﬂabellifolius1