Composition and desiccation-induced alterations of the cell wall in the

resurrection plant Craterostigma wilmsii

Maıte Vicrea, Olivier Lerouxela, Jill Farrantb, Patrice Lerougea and Azeddine Driouicha,*

aUMR CNRS 6037, IFRMP 23, Centre Commun de Microscopie Electronique. Universite de Rouen, 76821 Mont Saint Aignan Cedex, FrancebMolecular and Cell Biology Department University of Cape Town Private Bag, Rondebosch 7701 South Africa*Corresponding author, e-mail: azeddine.driouich@university-rouen.fr

Received 14 February 2003; revised 19 June 2003

Resurrection plants have the unique capacity to revive from an

air-dried state. In order to tolerate desiccation they have toovercome a number of stresses, mechanical stress being one.

In leaves of the Craterostigma species, an extensive shrinkage

occurs during drying as well as a considerable cell wall fold-

ing. Our previous microscopically analysis using immunocy-tochemistry on the resurrection plant Craterostigma wilmsii,has shown an increase in labelling of xyloglucan and unesteri-

fied pectins in the cell wall during drying. In this study, we haveundertaken a biochemical approach to separate, quantify and

characterize major cell wall polysaccharides in fully hydrated

and dry leaves of C.wilmsii. Our results show that the overall

cell wall composition of C.wilmsii leaves was similar to that of

other dicotyledonous plants with respect to the pectin content.

However, the structure of the hemicellulosic polysaccharidexyloglucan was characterized to be XXGG-type. The data

also demonstrate marked changes in the hemicellulosic wall

fraction from dry plants compared to hydrated ones. The most

conspicuous change was a decrease in glucose content in thehemicellulosic fraction of dry plants. In addition, xyloglucan

from the cell wall of dry leaves was relatively more substituted

with galactose than in hydrated walls. Together these findingsshow that dehydration induces significant alteration of poly-

saccharide content and structure in the cell wall of C.wilmsii,which in turn might be involved in the modulation of the

mechanical properties of the wall during dehydration.

Introduction

Over one third of the earth’s surface is considered to bearid or semi-arid because it is subjected to permanentdrought (Bewley and Krochko 1982). Plants found in dryareas have developed astonishing ways to survive waterdeficit stress. Water-deficit tolerance is a strategy inwhich plants are able to experience protoplasmic dehy-dration without permanent injury (Bewley and Krochko1982). Many lower order species (mosses and ferns) cantolerate tissue desiccation (Bewley and Krochko 1982,Oliver 1996, Oliver et al. 1998). In higher plants, whileseeds and pollen are frequently desiccation tolerant, thevegetative tissues rarely are. However, there are a fewangiosperms, termed resurrection plants (Gaff 1971) inwhich the vegetative tissues have the remarkable capacity

to withstand severe water loss. During dehydration,desiccation-tolerant plants have to ameliorate againstnumerous stresses as the water is gradually lost fromthe cells (Bartels et al. 1990, Tuba et al. 1994, Daceet al. 1998, Farrant 2000, Scott 2000, Kranner et al.2002). To do so, resurrection plants have evolved a numberof molecular mechanisms, including the biosynthesis ofLEA (late-embryogenesis abundant) proteins, or modific-ation of lipid composition and carbohydrates (reviewedin Vertucci and Farrant 1995, Ingram and Bartels 1996,Walters et al. 2002).

One of the first and major stress plants have to over-come in order to survive desiccation is mechanical stress(Iljin 1957). For some of the resurrection plants such

PHYSIOLOGIA PLANTARUM 120: 229–239. 2004 Copyright# Physiologia Plantarum 2004

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Abbreviations – AGP, arabinogalactan-protein;CWM, cell wallmaterial; CWR, cell wall residue;GC, gas chromatography;HG, homogalacturonan;MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MHDP, m-hydroxyphenyl method;RGI, rhamnogalacturonan I; RGII: rhamnogalacturonan II; TFA, trifluoroacetic acid; XG, xyloglucan.

Physiol. Plant. 120, 2004 229

Selaginella lepidophylla and the Craterostigma species,upon drying, the cell walls of vegetative tissues fold,becoming highly convoluted (Sherwin 1995, Thomsonand Platt 1997). When the plant is rehydrated, cells returnto their original volume without apparent injury. In theresurrection plant C.wilmsii a diminution of approxi-mately 78% of the initial cell volume occurs when theplant is dried (Farrant 2000). This reduction in cell size isdue almost entirely to an extensive folding of the cell wall.It has been proposed that the folding of the cell wall is astrategy developed by the cell to avoid the tearing of theplasmalemma from the cell wall during dehydration, thusallowing the cell integrity to be maintained (Farrant andSherwin 1997). Such folded cell walls are also a commonfeature in dry seeds and the manner of the cell wall collapseis characteristic of a given species (Webb andArnott 1982).These authors have suggested: (1) that wall folding in seedcells is essential for preserving the structural integrity of thetissue and retaining the viability of the seeds upon rehydra-tion; and (2) that the extent and the manner of cell wallfolding observed in various seed species depend upon cellwall chemical composition and structure.

The cell walls of plants are dynamic structures whichplay critical roles in plant morphology, growth anddevelopment (Albersheim et al. 1994, Penell 1998). Cellwalls are also fundamentally involved in mediating plantresponses to pathogenic and environmental stress (Zwiazek1991, Boudart et al. 1998). Despite this ascribed import-ance, our understanding of the function of plant cellwalls is still not complete. The recent characterizationof many Arabidopsis mutants with altered cell walls ispromising in providing a better understanding of thebiosynthesis and function of plant cell walls (Reiter et al.1997, Peng et al. 2000, His et al. 2001, Andeme-Onzighiet al. 2002). Nevertheless, resurrection plants such asCraterostigma species also provide an excellent system toexplore the function of the cell walls upon environmentalchanges. Many reports have illustrated the involvementof the cell wall in the response to varying environmentalfactors such as wounding (Cardemil and Riquelme1991), pathogen attacks (Boudart et al. 1998), coldacclimation (Weiser et al. 1990, Fujikawa et al. 1999,Kubacka-Zebalska and Kacperska 1999, Stefanowskaet al. 1999), osmotic stress (Hohl and Schopfer 1995,Wakabayashi et al. 1997, Marshall et al. 1999) salinestress (Iraki et al. 1989a, 1989b, 1989c) and dehydration(Zwiazek 1991, Ha et al. 1997).

To date there have been no studies on the character-ization of folded cell walls of the resurrection plantC.wilmsii upon drying, except our previous microscopi-cal analysis (Vicre et al. 1999). In that report, usingimmunocytochemistry, we studied the distribution ofxyloglucan (XG) and various pectic epitopes in the cellwalls of C.wilmsii leaves during drying and rehydration(Vicre et al. 1999). We showed that the cell wall foldinginduced by dehydration was accompanied by a markedincrease in XG and polygalacturonic acid/rhamnogalac-turonan I (PGA/RG-I) epitopes, with levels decliningagain during rehydration.

On the basis of these observations, we wanted tofurther analyse the chemical composition of C.wilmsiiwalls using biochemical methods. In the present study,we describe the fractionation and structural analysis ofpolysaccharides isolated from the cell walls of fullyhydrated and dehydrated leaves. This represents thefirst report on the cell wall chemical composition ofdesiccation-tolerant higher plants.

Materials and methods

Plant material

Plants of C.wilmsii (Scrophulareaceae) were collectedfrom Buffelskloof Nature Reserve, Mpumulanga, SouthAfrica and were maintained in a glasshouse at theUniversity of Cape Town as previously described(Sherwin and Farrant 1996). Whole plants were driedby withholding water and allowing the plant to drynaturally under ambient conditions for 15 days.

Sample preparations for scanning electron microscopy

(SEM)

Leaves from both hydrated and dry plants were cryo-fixed in liquid nitrogen slush and sublimed at �70�C(frozen hydrated samples). They were coated with goldpalladium and viewed using the cooled stage in a Hitachis-570 scanning electron microscope.

Sample preparations for transmission electron microscopy

(TEM)

Leaf fragments (1–2mm) were excised from the mid-blade of the plant rosette. These were fixed overnight in2.5% glutaraldehyde in 0.1M phosphate buffer (pH7.4)containing 0.5% caffeine (Sherwin and Farrant 1996).Post fixation was in 1% osmium tetroxide in phosphatebuffer. Following ethanol dehydration, the material wasembedded in epoxy resin (Spurr 1969). Thin sections(90 nm) were cut using a Reichtert Ultracut-S ultrami-crotome and collected onto 200m mesh copper grids.Sections were stained with uranyl acetate (10min) andlead citrate (10min) as previously described in Vicre et al.(1999) and were observed at 80 kV on a Zeiss transmis-sion electron microscope (EM 109).

Isolation of cell wall material (CWM)

Leaves from hydrated and dry plants were frozen inliquid N2. The freeze dried leaves (2 g) were ground toa powder in a mortar and suspended in boiling ethanol(1 g: 100ml) for 15min. After filtration, the residue wassuspended in 100 ml of methanol/acetone (1:1) underagitation for 24 h then washed thoroughly with 80%acetone. The isolated cell walls were filtered and allowedto dry in an oven at 80�C overnight. The weights and theyields were then determined.

230 Physiol. Plant. 120, 2004

Fractionation of CWM

CWM (dry powder) was extracted as follows: three timeswith water (500mg/150ml) at 100�C for 2 h each withconstant stirring, three times with 0.1% EDTA (pH7.5)at 100�C for 2 h each and once with 1.5M boiling NaOH(Girault et al. 2000). The NaOH solution contained 1%(w/v) NaBH4 and the extraction carried out under N2 withcontinuous stirring. The NaOH extract was neutralizedwith amberlite IRH resin. All fractions were filtered,dialysed against deionized water for 24h, lyophilized andweighed. The yield was determined for each fraction.The remaining cell wall residue (CWR) was dried andweighed, and sometimes hydrolysed with trifluoroaceticacid (TFA) in order to analyse themonosaccharide contentof non-cellulosic polysaccharides. All results expressedwere usually obtained from three experiments or more.

TFA hydrolysis of the remaining cell wall residue (CWR)

For further analysis of sugar composition determination,5mg of CWR was dissolved in 3ml of 2M TFA for30–60min at 100�C in sealed tubes. Then the supernatantwas dried under air stream and used for sugar analysis byGC as described below.

Sugar quantification

Total sugar content of each fraction was determined bythe phenol sulphuric method using galacturonic acid(Gal A) as standard (Dubois et al. 1956). Uronic acidcontent was determined by the m-hydroxyphenyl method(MHDP) according to Blumenkrantz and Absoe-Hansen 1973) using Gal A as a standard sugar. Allexperiments were performed 10 times.

Monosaccharide composition analysed by Gas

Chromatography (GC)

The sugar composition of cell wall fraction was determinedby gas chromatography analysis of their trimethyl-silylglycosides according to York et al. (1985). Samples(0.5mg of polysaccharide) were mixed with methanol/HCl 1M (500 ml) for methanolysis 24 h at 80�C. Inositolwas used as internal standard. The solution was thenevaporated under an airstream and the samples werewashed with methanol and dried. This step was repeatedthree times. Samples were methylsialylated with 100ml ofTri-Sil for 2 h at 80�C. The solution was evaporatedunder an air stream and the residue was suspended in20 ml of pyridine and 1ml of cyclohexane. One ml wasloaded on the injector and injected directly on a DB-1column (DB-1 Supelco). Chromatographic data wereintegrated with GC Star Workstation software (Varian),each surface being corrected according to its responsefactor. Sugars detected by GC were arabinose (Ara),fucose (Fuc), galactose (Gal), galacturonic acid (Gal A),glucose (Glc), glucuronic acid (Glc A), mannose (Man),rhamnose (Rha) and xylose (Xyl).

Detection of arabinogalactan-proteins (AGPs) with the

Yariv reagent

b-D-glucosyl Yariv reagent (Biosupplies, Australia), whichbinds and precipitates AGPs (Fincher et al. 1983) was usedto detect AGPs in different cell wall fractions. 1% agarosegel containing 0.15M NaCl and 30mgml�1 Yariv wasplaced in a Petri dish. Five ml of each fraction as well asAGP standards was loaded into a small well made in thegel. Diffusion was carried out at 37�C in a humid chamberfor 24h. Standards were made of a red wine purified AGP(Pellerin et al. 1995) and used in a range of concentrationbetween 0.2mgml�1 to 3mgml�1.

Mass spectrometry analysis of XG fragments

XG fragments were generated by treating 1mg of theNaOH fraction with 5U of endo-b-(1,4) glucanase (Mega-zyme International, Ireland, EC 3.2.1.4) in 500 ml of10mM AcNa for 18 h. Matrix-Assisted Laser DesorptionIonization-Time of Flight mass spectra (MALDI-TOFMS) of the resulting xyloglucan solubilized fragmentswere recorded on a Micromass (Manchester, UK) Tofspec E MALDI-TOF mass spectrometer. Mass spectrawere performed in the reflectron mode. This instrumentwas operated at an accelerating voltage of 20 kV with areflectron potential of 26 kV, and a pressure of approxi-mately 10�7 mbars in the source and 10�6 mbars in theanalyser. Samples were desorbed and ionized from theprobe tip with a nitrogen laser (l¼ 337 nm) with apulsewidth of 4 ns. More than 12 laser shots were sumsfor each mass spectrum. Two ml of the endoglucanase-generated-xyloglucan oligosaccharides solution wasdissolved in the same volume of matrix prepared by dis-solution of 2mg of 2, 5-dihydroxybenzoic acid in 200ml of70% (v/v) acetonitrile in 0.1% (v/v) TFA. The sample-matrix mixture obtained was homogenized and 2ml ofthis solution was deposited onto probe tips and allow todry for a few minutes under vacuum. The target was thenapplied in the MALDI-TOF mass spectrometer.

Monosaccharides content of XG fragments

XG fragments after enzymatic digestion were purified byusing a carbograph (Altech) column (Packer et al. 1998).A carbograph column was prewashed before use with5ml acetonitrile 90% with 0.1% TFA and by 5ml deio-nized water. The samples were placed on the graphitecolumn and washed with 5ml water. Neutral oligosac-charides, like XG fragments, were selectively eluted using5ml of acetonitrile 25%. This fraction was then collected,freeze-dried, treated with MeOH/HCl and silylationreagent for GC analysis (York et al. 1985).

Results

Morphology of dry plants

The morphological changes undergone by the resurrec-tion plant C.wilmsii during drying are presented in

Physiol. Plant. 120, 2004 231

Fig. 1. In fully hydrated plants, the leaves were green andexpanded (Fig. 1A). As the plant dried, leaves curledinward and a purple colour characteristic of anthocyaninpigments was visible on the abaxial surface. In the drystate the leaves were tightly folded with abaxial surfacesof the outermost leaves exposed to sunlight (Fig. 1B).

Microscopical study

Figure 2A-D show cryo-scanning electron micrographsof hydrated and dry leaves. In transverse sections fromhydrated leaves, cells were characterized by a roundedshape in both spongy and palisade parenchyma cells(Fig. 2A). In contrast, cells in dried leaves were foldedand highly shrunken (Fig. 2B). It is interesting to notethat cell wall undulations were apparent in the threedimensions without any regular pattern or orientation(Fig. 2B). Only xylem, being more rigid, maintained itsoriginal shape. The boundary between palisade andspongy parenchyma was no longer discernible and thesurface of lower epidermis (abaxial surface) exhibitednumerous hairs. This is the face exposed to the lightwhen the plant is dried and we assume that their role isessentially a protection against light stress.

Figure 2C,D show surface views of epidermis fromhydrated and dry tissues, respectively. In Fig. 2C stomataand glands are visible. Epidermis of dry leaves appearedconvoluted and highly folded (Fig. 2D). It is worthnoting that stomata of dried leaves were always open.Schwab et al. (1989) reported that in Craterostigmaplantagagineum, drying results first in a closure of thestomata, but when the water potential drops further theycould be passively reopened. This may also be the casewith C.wilmsii, and tensions created as the epidermisfolds could lead to a passive reopening of stomata.

The ultrastructure of chemically fixed hydrated anddry leaf cells is shown in Fig. 3A,B. In hydrated leafcells, the cytoplasm, including organelles such as chloro-plasts were peripherally located along the cell wall, withnormal organization (Fig. 3A). The cell wall was alsonormal in appearance and a large vacuole, which com-pletely fills the cell, was present. In the cells of driedleaves the cytoplasm occupied the cell volume as thecentral vacuole disappeared and considerable cell wallfolding was observed (Fig. 3B). Most of the chloroplastswere rounded in shape and were localized in the middleof the cell. Such protoplasmic dehydration-inducedalterations of the cellular anatomy have been previouslyreported in C. plantagineum by Schneider et al. (1993).Note that the thylakoid membranes were well defined indry cells and the plasmalemma was not always immedi-ately adjacent to the folded cell walls, with some with-drawal evident in some cells.

We have previously demonstrated that folded cellwalls in dehydrated leaves of C.wilmsii presented signifi-cant alterations of XG and some pectin epitopes (Vicreet al. 1999). Therefore, we wanted to gain better infor-mation on the precise chemical composition of cell wallsof hydrated and dry C.wilmsii plants.

Yield and total sugar content of cell wall fractions

Cell walls of hydrated and dried cells were isolated andpolysaccharides sequentially extracted with hot water(HW), the calcium chelator EDTA and 1.5M NaOH(see Materials and methods). The total amount of the cellwall isolated from hydrated leaf cells was quite similar tothat of the walls from dried plants (317 versus 350mgg�1

of lyophilized leaves). As shown in Table 1, the yield of thematerial extracted from the cell wall of hydrated plants

A B

Fig. 1. Craterostigma wilmsii plants. (A) Fully hydrated plants have green expanded leaves; (B) dried plants have curling leaves, only olderleaves are exposed to the sunlight. Note the purple colour of dried leaves, indicating the presence of anthocyanin on the abaxial surface.

232 Physiol. Plant. 120, 2004

represented 15, 28 and 37%, in HW, EDTA and NaOHfractions, respectively, and was not significantly differentfrom that of dried plants.

In contrast, dehydration resulted in a marked increasein the neutral sugars in the EDTA fraction, whereas onlya slight decrease was detected in HW and NaOH frac-tions. Similarly, uronic acid content exhibited a 2-foldincrease in the EDTA fraction of dehydrated plants.Nevertheless, it is worth noting that polysaccharide frac-tions extracted from dry plant cell walls were much moreeasily solubilized in water than those of hydrated ones.

Chemical composition of cell walls

The composition of cell walls isolated from hydrated anddried plants is presented in Table 2.

The amount of each monosaccharide in the three frac-tions as well as in the remaining cell wall residue (CWR)was determined using GC.

Hot water extract The major sugars found in HW solublematerial are characteristic of pectins. In both hydratedand dry plants, the HW cell wall fraction was mainlycomposed of Gal A, Ara and Gal although Glc, Glc A

A B

DC

ue

pp

vs

sp

le

g

st

st

h

le

vs

ue

sp

pp

h

Fig. 2. Cryoscanning electron micrographs of hydrated and dried C.wilmsii leaves. (A-B) Transverse sections of hydrated (A) and driedC.wilmsii leaves (B). (C-D) Surface view of the epidermis of hydrated (C) and dried (D) leaves. Glands are present in the epidermis. Note thatin the dry leaves, the stomata are largely opened. g, gland; h, hair; le, lower epidermis; st, stomata; sp, spongy parenchyma; pp, palissadicparenchyma; ue, upper epidermis; vs, vessels; !, cell wall folding. Bars: a, b¼ 200mm; c,d¼ 40mm.

Physiol. Plant. 120, 2004 233

and small amounts of Rha, Xyl and Man were alsopresent. Gal A was the predominant sugar in both cellwalls, accounting for 32.1+5% and 38.9+3.7% of totalsugars from hydrated and dry plants, respectively. Nomajor difference in the content of various sugars wasdetected. Although the sugar composition of this frac-tion was indicative of pectins, some AGPs were alsopresent, as revealed by gel diffusion and Yariv staining(Fig. 4).

EDTA extract The sugar composition of chelator-soluble fractions comprised mainly Gal A, in both hydrated(61.3+5.9%) and dry (60.2+6.1%) plants (Table 2).Small amounts of Gal and Ara were also detected, butall the other sugars were present in very minor propor-tions. The content of sugars was not significantly differ-ent between dry and hydrated plants. In addition, as forHW fractions, gel diffusion and Yariv staining revealedthe presence of AGPs in the EDTA fractions for bothhydrated and dry plants (Fig. 4).

1.5 M NaOH extract In both hydrated and dry plant cellwalls, the alkali-soluble fraction represented the mainfraction accounting for about 37% (Table 1). The majorsugars detected were Glc, Gal and Xyl in both hydratedand dry plants demonstrating the hemicellulosic natureof this fraction and suggesting the presence of XG. Inhydrated plants, Glc was the predominant neutral sugarin the NaOH fraction representing 30.5+3.1%, whereasGal and Xyl represented 17.4+3% and 16.2+3%,respectively. Interestingly, the content of the predomi-nant sugar, Glc, was reduced by approximately 2-fold indry plants (17.7+1.9%). Another notable finding of thisanalysis is that, unlike Glc, Gal was lower in hydratedthan in dry cell walls. The presence of high levels of GalA indicates that some pectins were also solubilized by thealkali treatment.

CWR after TFA hydrolysis Although the CWR left aftersequential extraction by HW, EDTA and NaOH wasexpected to contain mainly cellulose, the analysis of itsmonosaccharide contents has shown that non-cellulosicpolysaccharides were still present in this fraction. TheCWR fraction was treated with 2N TFA to hydrolyseall polysaccharides except cellulose, and sugar composi-tion was determined using GC (Table 2). The presence ofrelatively high levels of Glc, Gal and Xyl suggests thatXG was still present in the CWR. This was probablytightly bound to cellulose. Again the Glc content wasreduced in dry plants as compared to hydrated ones,confirming the result obtained with the NaOH fraction.Interestingly, the contents of both Xyl and Gal werehigher than Glc content in the cell walls of dry plants.As Glc is usually the predominant sugar in XG, thisindicates that Xyl and Gal may originate from otherpolymers than XG or that XG from dry plants evolveda different structure and sugar composition.

Determination of XG structure

To analyse further the change in the Glc content of theNaOH fraction, we focused on XG, the major hemicel-lulosic polysaccharide in primary walls of dicot plants,which is the main source of Glc in this fraction. Todetermine the XG structure, the NaOH soluble fractionwas digested with endoglucanase to generate specificXG-fragments that were analysed by MALDI-TOF massspectrometry. For hydrated and dry plants, three mainions were obtained which corresponded to XG-derived

cyt

cyt

cp

v

cw

cp

A

B

cw

v

Fig. 3. Transmission electron micrographs of transverse section ofhydrated (A) and dry (B) leaves of C.wilmsii fixed by conventionalfixation. In hydrated leaves, cells have a rounded shape and thecytoplasm forms a thin layer close to the cell wall. In dry cells, cellwalls folding is clearly seen (arrows). cp, chloroplast; cw, cell wall;cyt, cytoplasm; v, vacuole; !, cell wall folding. Bars: a,b¼ 5mm.

234 Physiol. Plant. 120, 2004

oligosaccharides and were composed only of three to fivehexoses, interestingly with only two pentoses (Fig. 5).This MALDI-TOF analysis enabled us to concludefirstly that the XG of C.wilmsii is not fucosylated, asthe mass spectra did not show any ion compatible withstructure containing a deoxy-hexose. Secondly, the corestructure of this XG appeared to be of a XXGG-typeaccording to the nomenclature of Fry et al. (1993). Thus,to determine the monosaccharide content and check forthe presence of Gal, XG fragments were purified on acarbograph column and analysed by GC. These XGfragments were mainly composed of Glc, Xyl and Gal,comprising about 90% of the total sugar, in the ratio of(4:1.5:0.8) and (4:2.5:2) for hydrated and dry plants,respectively (Fig. 6). Minor amounts of Man were alsodetected, suggesting the presence of glucomannan.

Discussion

The result of this study demonstrates that: (1) leaf cellwalls of C.wilmsii contain the same major polysacchar-ides found in the primary cell walls of many dicot plants;(2) that the hemicellulosic polysaccharide XG has a par-

ticular structure with no Fuc substitutions like XG fromSolanaceous plants (Vincken et al. 1997) and the storageXG of tamarind seeds (York et al. 1993); and (3) that thecontent and structure of certain matrix polysaccharidessuch as XG was altered during dehydration.

Overall composition of C.wilmsii cell wall

The carbohydrate components of C.wilmsii cell wallswere separated into four fractions, the water, EDTA,and NaOH-soluble fractions, as well as the remainingcell wall residue. Leaf cell walls of C.wilmsii were char-acterized by a large proportion of homogalacturonans asjudged from the EDTA fraction which contained up to60% Gal A. This fraction represents around 30% of leafcell walls twice that of the water fraction. Similarly,sugar composition of water-soluble polysaccharidesrevealed Gal A as a major sugar (around 30%) indicativeof the presence of homogalacturonan (HG). Accordingto the classification of pectins by Schols and Voragen(1996), which is based in part on the ratio of Rha to Gal A,rhamnogalacturonans are differentiated from homogalac-turonans by having a ratio Rha/Gal A varying between

Table 1. Amount of the cell wall material and carbohydrates solubilized with hot water, EDTA and NaOH from hydrated and dry plants.Yields are expressed as weight percent of the total cell wall material. Assays for total sugars and uronic acid were repeated 10 and 6 times,respectively*. Values are expressed in mgmg�1 cell wall. Protein content was estimated according to Bradford assay.

Hydrated plants Dry plants

Cell wall(mg g�1 lyophilized leaves)

317 350

Hot waterYield (W/W) 15% 15%Total sugars* 128+10 110+16Uronic acids* 42+12 36+7Proteins 4.3 5.6

EDTAYield (W/W) 28% 32%Total sugars* 140+38 259+29Uronic acids* 50+25 102+25Proteins 5 4.8

NaOHYield (W/W) 37% 36%Total sugars* 122+22 97+14Proteins 6.3 4.7

Residue (CWR)Yield (W/W) 20% 17%

Table 2. Monosaccharide composition of the cell wall fractions from hydrated (H) and dry (D) C.wilmsii leaves. Values are mol percentage ofthe sugar recovered. Data are means of at least 3-values. * Sugars solubilized from residual material by TFA, does not include cellulose.

Hot water fraction EDTA fraction NaOH fraction Cell wall residue *

H D H D H D H D

Ara 16.5+4.9 25.7+7.7 12.4+1.8 9.2+4.5 8.7+0.6 9.6+2 3.9+3.4 1.6+1.5Rha 5.0+0.3 5.2+3.2 5.8+0.6 6.4+1.5 3.4+1.0 3.9+0.8 0.8+0.1 0.7+0.2Xyl 3.1+1.6 2.8+1.1 4.8+0.7 3.5+2.0 16.2+3.0 17.7+1.5 19.9+1.0 34.2+5.3Man 1.7+1.1 2.5+0.3 0.9+0.1 0.6+0.2 7.2+1.0 5.0+1.0 1.7+0.3 3.2+0.4Gal 18.1+11.2 11.7+1.3 9.7+2.4 14.6+2.6 17.4+2.9 24.2+1.9 13.1+4.0 35.5+3.3Glc 11.8+3.7 7.2+1.7 3.1+1.6 3.1+0.7 30.5+3.1 17.7+1.9 49.2+9.5 18.7+2.1GlcA 14.0+6.9 5.7+4.9 1.6+0.4 2.2+1.1 3.5+2.2 4.3+3.5 1.6+1.7 1.3+0.5Gal A 32.1+5 38.9+3.7 61.3+5.9 60.2+6.1 13.1+1.8 17.4+1.6 11.8+4.3 4.7+3.7

Physiol. Plant. 120, 2004 235

0.05 and 1. The Rha to Gal A ratio in the water fractionranged between 0.13 and 0.16 for hydrated and dryplants, indicating that rhamnogalacturonan was alsopresent. Calculation of this ratio for the EDTA fractionindicates the presence of the polysaccharide rhamnoga-lacturonan I (RG I), probably in lower amounts than inthe water fraction. The presence of Ara and Gal in bothfractions suggests the occurrence of these sugars on sidechains of RG I. However, these residues may reflect thepresence of arabinogalactan or arabinogalactan-proteins,the latter being detected by Yariv staining on agarose gelin both fractions (Fig. 4). Glycosyl-residue compositionof the alkali-soluble fraction showed that it containspredominantly Glc, Xyl and Gal, which are the primarysugars of XG of primary walls (Bacic et al. 1988). Thus,as in all dicot plants, XG seems to be the major hemi-

cellulosic polysaccharide in C.wilmsii leaf cell walls.However, other hemicelluloses such as xylans or gluco-mannans might also be present. In addition, xyloglucan-like polysaccharide was released upon TFA treatment ofthe cell wall residue, indicating that some XG in C.wilmsiiwalls was also tightly bound to cellulose. However, inthis study we did not check for the presence of rhamno-galacturonan II (RG II). Nevertheless, using anti-RG IIantibodies (Matoh et al. 1998) and immunocytochemistrywe could detect this polysaccharide in the cell walls ofC.wilmsii leaves (Vicre 2001).

Desiccation-induced alterations of cell wall

polysaccharides

In a previous immunocytochemical study, we showedthat dehydration of C.wilmsii was accompanied by cellwall folding and alteration of XG and pectin epitopescontent. No such changes were observed in a controlnon-tolerant plant Pisum sativum (Vicre et al. 1999). Inthe present study, we found that the total sugars anduronic acids content in the EDTA soluble fraction wasmuch higher in dry than in hydrated plants (Table 1),which is suggestive of a possible increase in pectins. Thisincrease would explain the increase in the immunogold-labelling of acidic pectins we previously observed (Vicreet al. 1999). However, a complete solubilization of EDTAfraction prepared from hydrated leaves was always diffi-cult to achieve. Attempts to further solubilize these frac-tions with TFA significantly reduced the difference in thecontent of total sugars between hydrated and dry plants.Thus the difference in uronic acid observed could beattributed to modifications of pectin solubilization prop-erties rather than specific changes in the amount of thesepolysaccharides. This is interesting as it reflects changesin the reorganization and may be bonding of pectins toother wall polymers, which may occur upon desiccation.Such changes would affect the overall architecture of thecell wall, thereby contributing to the observed folding(i.e. extensibility) and alterations in the level of pectic

Hot water EDTA NaOH

H H HD DD

AGP purified from red wine (mg/ml)

0.2 0.4 1 2 3

Fig. 4. Arabinogalactan proteins(AGPs) detection in different cellwall fractions solubilized fromhydrated (H) and dry (D) plantsby Yariv on agarose gel (lowerspots). Red spots are indicative ofthe presence of AGPs in differentfractions. A red wine purifiedAGP was used at differentconcentrations as a standard(upper spots).

Fig. 5. MALDI-TOF mass spectra of the xyloglucan of the NaOHfraction solubilized from the walls of hydrated (A) and dry (B)leaves of C.wilmsii. Xyloglucan fragments (Hex3�6 Pent2) weregenerated by treatment with endoglucanase (see Materials andmethods). Hex, hexose; Pent, pentose.

236 Physiol. Plant. 120, 2004

epitopes detected immunocytochemically (Vicre et al.1999). Such an increase in the solubility of pectinswas reported following environmental stress or hormonetreatment (Terry et al. 1982, Iraki et al. 1989b) andsuggested to accompany increases in the rate of wallexpansion.

Another striking difference in sugar composition isthat Glc content of the alkali-soluble fraction was muchlower in dry plants than in hydrated ones. This was alsothe case for the TFA-soluble fraction after hydrolysis ofthe cellulose-rich residue. In theory, any starch presentshould be solubilized at the hot water stage, and treat-ment of the NaOH soluble fraction with a-amylase didnot reveal the presence of starch (data not shown). Thelower content of Glc in the NaOH fraction could be dueto a modification of the XG during dehydration. Struc-tural analysis of this polysaccharide revealed that it isof a XXGG-type (Fry et al. 1993) which is typical of XGof Solanaceous plants (Vincken et al. 1997). Analysis ofmass spectra of the XG fragments does not show anyqualitative difference of the three main ions observed(Fig. 5), between hydrated and dry plants. In order todetermine if the modification of Glc content of theNaOH fraction was associated with XG or due to acontamination of other polysaccharides, the same XGfragments as those used for MALDI-TOF were purifiedon carbograph column analysed by GC. Purified XGfragments of dry plants had a relatively lower contentof Glc consistent with what was observed for the globalanalysis of the NaOH fraction. Although we cannotexclude a contamination of the hydrated NaOH fractionby other polysaccharides such as glucomannan, this datacould suggest a possible XG trimming during dehydra-tion, with a particular removal of poorly substituteddomains of this polymer. As a consequence, XG ofhydrated plants, with a higher proportion of less substi-tuted domains, would appear to be richer in Glc contentcompared to its Xyl and Gal contents. If this were thecase, cleavage and/or partial degradation of XG could beable to modify the mechanical property of C.wilmsii cellwalls, increasing its elasticity in response to dehydration.It is worth noting that Sherwin (1995) showed that the

leaves of Craterostigma nanum became more elasticduring dehydration. Considering the large degree ofshrinkage in cell volumes and the great capacity of thewall to fold, it seems reasonable to expect the cell walls ofleaves of C.wilmsii to be remarkably elastic duringdrying in order to prevent any irreversible damage.Furthermore, changes in XG have previously beenreported to play a role in many plant species in responseto water stress. Zwiazek (1991) indicated that whitespruce needles had increased hemicellulose content afterpreconditioning treatment or severe drought stress expo-sure and Kubacka-Zebalska and Kacperska (1999) foundthat the hemicellulose fraction of cold acclimated leavesof winter oilseed rape subjected to frost treatment haddecreased Glc and Xyl contents.

Sugar metabolism plays a major role in desiccationtolerance of C. plantagineum (Bianchi et al. 1992, Ingramet al. 1997, Norwood et al. 1999, 2000). This resurrectionplant accumulates a large amount of octulose (a raresugar in higher plants) in fully hydrated leaves tissues,whereas upon dehydration octulose level decreases andsucrose concentration increases up to 90% of the totalsugar content (Bianchi et al. 1992). It would appear thatCraterostigma species have evolved a capacity to accu-mulate sucrose very rapidly from carbohydrate sourcesalready present in the leaf. In the case of C.wilmsii, oneof these carbohydrate sources could be the cell wall, andmore particularly XG. Thus, an additional explanationfor the loss of Glc in dry plants would be that Glc is usedfor sucrose synthesis. The Glc released either from thecell walls or starch might be an important energy sourcewithin the cytoplasm, for the induction of protectionmechanisms involved in desiccation tolerance in this species.

Acknowledgements – We are grateful to Dr Girault for his help ininitiating the biochemical analysis of the cell wall of C.wilmsii atthe University of Rouen. We would like to thank Drs B. White,J. Mesjaz-Przybylowicz and W. J. Przybylowicz for their help duringthe cryoworkshop in Durban (1998) and John and Sandie Burrows forplant collection from Buffelskloof Nature Reserve. Work at UCT wassupported by grants to J.F. from the National Research foundationand UCT University Research Committee. Work at A.D labora-tory was supported by l’Universite de Rouen, le Conseil Regionalde Haute Normandie and le Ministere des Affaires Etrangeres.

Monosaccharides content of XG fragments

0

10

20

30

40

50

60

Ara Rha Xyl GalA GlcA Man Gal Glc

Mo

lar

per

cen

tag

e (m

ol %

)

HydratedDry

Fig. 6. Monosaccharide compos-ition of the endoglucanase-generated xyloglucan fragmentsobtained from the NaOH fractionsolubilized from the cell wall ofhydrated and dry leaves ofC.wilmsii.

Physiol. Plant. 120, 2004 237

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Edited by C. Guy

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