J. Cell Sd. 33, 325-336 (1978) 325Printed in Great Britain © Company of Biologists Limited IQJS

CELL WALL DIFFERENTIATION AND

STAGES INVOLVED WITH INTERCELLULAR

GAS SPACE OPENING

J. C. ROLANDLaboratoire de Biologie Vig&tale - Cytologie Expe'rimentale,Umversite de Paris, ENS, 24 rue Lhomond, 75231 Paris Cedex 05, France

SUMMARY

The development of the intercellular gas system has been followed during the growth ofPisum sativum root and Phaseolus aureus hypocotyl by means of ultrastructural cytochemistry.

The extension of the system is sequential and takes place according to a defined programmeof cell wall reconstruction. Contrary to current views, the actual pectic middle lamella doesnot seem to be directly engaged in the initiation of the aerating system, which converselyappears subordinate to the presence of specialized substructures within the wall. The processis characterized by the early differentiation of a particular layer of wall called the 'splitting layer'.The splitting layer differs from the pectic middle lamella particularly in its insolubility in anincubating medium which removes the wall subunits (EDTA, DMSO, pectinases, cellulases)and its non-reactivity to polysaccharide test involving periodic oxidation (PATAg staining).With ultracryotomy, it displays a distinctive yS-glycerophosphatase activity.

The layer gradually splits apart from lateral sites in a manner which somewhat evokes theopening of a zip fastener. The primordial opening, and later the intercellular space, keeps athin (10-20 nm) extramural coat which is apparently non-glucidic and derives from the splittinglayer. Ultimately, local shifts and resorptions of the wall lead to fusion of the early intercellularchannels.

One of the peculiarities of the opening of the air-space is that because of the mechanisminvolved the polysaccharides of the wall are not left naked. The processes observed are comparedwith other cases of cell wall separation.

INTRODUCTION

The cell wall has to ensure intercellular exchanges in plant tissues and from thispoint of view is somewhat comparable to the ' internal medium' of animals. Differentkinds of communications are regulated via the wall and liquid exchanges through theapoplasm and the symplasm (plasmodesmata) have been specially documented recently(see Gunning & Robards, 1976). The development of a gas system between cells hasnot received so much attention despite the importance of gas exchanges in plants.Knowledge about opening and growth of intercellular spaces is based on classicallight-microscopy studies (see Martens, 1937, 1938; McPherson, 1939; Biirstrom,1959, and reviews of Sifton 1945, 1957)-

Cytochemistry at the ultrastructural level makes it possible to define the organizationof the cell walls. The purpose of the present study was to follow by this means thesequence of events in formation of the 'plant respiratory system' during the courseof cell differentiation.

326 J. C. Roland

MATERIAL AND METHODS

Cortical cells of Pisum sativum roots and Phaseolus aureus hypocotyls in which primary walfine structure has been previously studied (Roland, Vian & Reis, 1975, 1977) were used. Thematerial was obtained from 4-day-old seedlings of pea and mung bean grown on wet vermiculitein the dark at 26 °C.

For conventional embedding in plastic the specimens were fixed in 3 % glutaraldehyde in0-2 M cacodylate buffer, pH 7-2, for 90 min at room temperature. After washing in buffer, theywere postfixed for 30 min in 1 % osmium tetroxide in the same buffer. After dehydration throughan ethanol series they were embedded in an Epon-Araldite mixture. Thick sections for surveyin light microscopy were stained with toluidine blue and/or periodic acid-Schiff. Thin sectionswere routinely stained with uranyl acetate-lead citrate.

Cytochemistry was performed with periodic acid-thiocarbohydrazide-silver proteinate(PATAg) for polysaccharides according to Thie'ry (1967). Conjunction of cytochemical stainingwith extractions of wall components was according to Reis & Roland (1974) and Freudlich &Robards (1974). For this purpose specimens, fixed with glutaraldehyde and washed, wereimmersed and stirred: (1) at room temperature overnight in dimethylsulphoxide (DMSO) or1 % aqueous ethylenediaminetetra-acetic acid (EDTA). DMSO and EDTA are solvents cur-rently used in carbohydrate biochemistry to extract respectively pectins and xylan-rich fractionof hemicelluloses (Thornber & Northcote, 1962; and, for the present material, Reis & Roland,1974, and Reis, 1974). Compared with other solvents used for polysaccharide extraction (alkali,etc.) they have the advantage of being more selective and causing less damage to cell substructure;(2) 1-6 h at 37 °C in 1 % cellulase (Onozuka) and/or 1 % pectinase (Macerozyme). Afterwashing, specimens were postfixed in osmium tetroxide 1 % and embedded as above. Stainingwith PATAg was performed subsequently on sections.

For acid phosphatase detection, incubation in Gomori medium with 8 mM/9-glycerophosphateas substrate in 0-05 M acetate buffer, pH 5-5 (Miller & Palade, 1964; Poux, 1970), was performedeither conventionally on tissue pieces or on sections. In the latter case, in order to avoid theproblem of penetration of substrate into the embedding medium, cryosections of non-embeddedspecimens were used.

Ultracryotomy was performed as described elsewhere (Roland, 1978) on specimens fixedbriefly (15 min) with glutaraldehyde and frozen in liquid nitrogen. Sections were cut at — 75 °Cwith a Sorvall MT2B microtome equipped with a cryokit FTS. After thawing at room tem-perature, the sections were floated 10 min — 2 h on the Gomori medium. Controls were incubatedwithout substrate or in presence of o-oi M sodium fluoride.

Abbreviations on figuresacecer

8VI

mln

amyloplastcorner reinforcementextramural coatendoplasmic reticulumGolgi bodymitochondriamiddle lamellanucleus

op

PissiV

distal opening in the splitting layer(= lateral site of commencement of split)plasm alemmaintercellular gas channelsplitting layervacuoleprimary wall

All sections are transverse except Fig. 2.

RESULTS

In both root tip and mung bean hypocotyl, the intercellular spaces in young anddifferentiating parenchyma formed individual channels regularly located at the cornersof cells within the longitudinal wall. They are thus parallel to the growth axis of theorgans (Figs. 1, 2). The tiers of cells are well oriented and. channel development can befollowed by means of serial sections through the youngest parts of the organs, i.e. in

Intercellular gas space opening 327

the last 2 mm of the root apex and in the hook of the hypocotyl where the intercalarymeristem responsible for elongation is located.

The intercellular space progresses regularly between the immature and still-dividing cells, in continuity with the pre-existing system. The sub-apical meristemof the pea root shows no intercellular spaces and no special intercellular substructurein its wall (Fig. 3). When the cells leave the meristematic state the opening of theintercellular spaces occurs very rapidly. While the vacuoles are still small and in thecourse of fusion, the opening is achieved at each corner of the cell (Figs. 1, 4). Thealterations of the wall related to air-space formation are one of the first cytologicallyevident indications that cells are engaged in the process of differentiation.

In vivo observation shows that the intercellular channels are already filled with gasat the beginning of the elongating zone. When the living root tip is placed on a slideunder a coverslip, the gas filling the extremities of the intercellular channel can beseen to dissolve instantaneously if a drop of KOH is added to the margin of the cover-slip. This suggests, as was stated by Burstrom (1959) for wheat root, that the gasinitially contained in immature tissues is essentially carbon dioxide.

Despite the rapidity of the process, successive events can be distinguished in thewall at the beginning of the elongation zone. Early stages are marked by the deposition,at the cellular junctions, of a special wall layer which, in view of its further evolution,will be called the 'splitting layer'. This layer is rather difficult to observe after routinestaining or directly after polysaccharide staining because of the high density andcompactness of the wall. Conversely, the splitting layer appears clearly when thetissue is incubated in a medium which removes to a greater or lesser extent the wallsub-units (EDTA, DMSO, pectinases or cellulases). Fig. 5 shows for example thesplitting layer visualized after a mild extraction of the wall with EDTA. Resistant toglycolytic treatments, the splitting layer appears distinct from the pectic middle lamellawhich is, on the contrary, easily removed with solvents or enzymes. The splittinglayer is resistant even to incubation in mixtures of pectinase and cellulase. It appearsthen well defined against the light background of the wall (Figs. 7, 8). However, afterlong enzymic treatments (6 h) the splitting layer becomes brittle and fragmented, asif it contained resistant components which are slowly removed. The layer extendsunequally from the cell junction to the cell facets. Each end of the cell is capped witha polysaccharide thickening. Along the layer, the middle lamella persists and showsno modification detectable with the PATAg staining. It simply divides at the celljunctions and lies beneath each side of the splitting layer. It can be noticed that thepectic lamella ceases there to be ' middle' or to form the party wall between cells.

After incubation of specimens en bloc in the Gomori medium for acid phosphatase,a positive reaction appears at the median part of the cell junction, with rather diffuselimits. Conversely, after ultracryotomy, when cryosections are floated on the samemedium the deposits of lead phosphate correspond exactly to the splitting layer (Fig.6). Neither the primary wall nor the middle lamella show lytic activity.

The deposition of the splitting layer is followed by its cleavage and the drawingapart of the adjacent walls. Characteristically the site at which opening begins islateral (Figs. 7, 8). The first minute chinks then progress gradually in a way which

J. C. Roland

Figs. 1-8 and 13-17 are cortical parenchyma at the beginning of the elongation zone,pea root. Figs. 9-12 are cortical parenchyma at the same point in mung bean hypocotyl.Figs. 1-4. General view of intercellular channels. Pea. PATAg staining; no extraction.

Fig. 1. Beginning of the elongation zone: vacuoles (u) still small and numerous;plastids with small starch granules (a). The intercellular channels (J) are already largelyopened at each angle of the cell. Arrow: new wall formed without direct relationshipwith the previously opened gas system, x 4700.

Fig. 2. Same region in longitudinal section showing the continuity of the inter-cellular channel (J). X 5200.

Fig. 3. Tricellular junction in the subapical root meristem. x 22500.Fig. 4. Same region at the beginning of the elongation zone, x 22 500.

Intercellular gas space opening 329

recalls the opening of a zip fastener. As soon as the intramural cavity is opened, itappears coated with a thin (10-20 nm thick) but continuous layer derived from thesplitting layer. This ' extramural coat' does not react to PATAg staining for poly-saccharides. The wall thickening capping the extremity of the cavity remains (Figs.7-9). It will form a 'corner reinforcement' limiting the intercellular channel (Fig. 14).

Figs. 5, 6. Individuality and lytic activity of the splitting layer. Sections near thesubapical meristem. Pea.

Fig. 5. PATAg staining + EDTA. The extraction of pectins reveals the insolublesplitting layer (si) within the wall (compare to Fig. 3). No cleavage intervenes yet.wi, remainder of the primary wall; c, future corner reinforcement, x 38000.

Fig. 6. Ultracryotomy (cryosection floated 1 h on Gomori medium for acid ̂ -glycero-phosphatase). The lead phosphate precipitates (arrows) correspond exactly to the siteof the splitting layer, x 38000.

In the corner reinforcement different components seem to be associated, some stronglyreactive to PATAg staining and easily extractable with glycolytic enzymes or solvents^others remaining after a glycolytic attack and poorly reactive to PATAg staining.Neither swelling of the middle lamella nor evidence of dispersion of microfibrillarmaterial have been seen during this stage of cleavage.

J. C. Roland

w1

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Intercellular gas space opening 331

A comparable process occurs in mung bean hypocotyl. In particular, the differentia-tion of a special wall layer associated with cleavage of the wall and opening of inter-cellular gas spaces is clearly visible. Here the extension is basifugal (towards the apexand the hook of the hypocotyl). The opening progresses often simultaneously fromthe axial and lateral parts of the splitting layer (Figs. 9-12). The cleavage extendsstep by step as described above. Early distinction between the splitting layer andmiddle lamella is shown on Fig. 9 after an extraction with DMSO. After cleavage,portions of the separated cell walls retain a thin non-reactive extraneous coat (Fig. 10,encircled area). Fig. 11 shows that in a given cell junction, the opening process is notequal in every direction; only certain facets of the cell wall are concerned and differen-tiate a splitting layer.

Later, the intercellular system can extend by fusion or anastomosis of intercellularchannels previously formed. Enlargement occurs as the result of a process of wallmodifications including local tearing and disintegration. The wall portion locatedbetween 2 neighbouring channels is gradually shifted and resorbed. In the lacunaformed, disrupted and more or less residual partitions indicate the former division(Figs. 15-17). In Fig. 13 both splitting and anastomosing occur in the same wall. Thelatter stage involving evident breakage and shift of the wall is probably what lightmicroscopists have described as air-space formation. In fact, it is only an ultimatestage of the air-space differentiation process and it should not be confused with theactual opening of intra-wall cavities.

DISCUSSION

Various kinds of intercellular spaces exist in plants (see for example: Esau, 1964;Morisset, 1968; Sifton, 1945, 1957). The extension of the intercellular gas system isusually considered to result directly from the disappearance of the pectic middlelamella. The most frequently cited references in this field are the studies of Martens(1937, 1938) who, following Jungers (1937), made classical observations on primaryand secondary tissues. The interpretation was that the pattern of the intercellularcavity is established at the time of cell division in the new partitioning wall, before itmeets the sides of the walls of the mother cell. When the cell divides, the free ends ofthe new middle lamella thicken and swell. This mass of wall enlarges and becomes a

Figs. 7, 8. Early opening of the splitting layer. Pea. PATAg staining + incubation 6 hin macerozyme and cellulase. Non-digestion of the splitting layer (il) whereas theothers wall components are removed, op, lateral site of commencement of cleavage inthe layer. Note the thickness of future intercellular corners (c). A thin non-reactiveextramural coat (ec, encircled area) is visible around the opening (op). Figs. 7, 8,x 32000 and x 80000, respectively.

Figs. 9, 10. Cleavage of the intramural cavity in mung bean hypocotyl. PATAgstaining + DMSO.

Fig. 9. Before cleavage. Non-reactivity of the splitting layer (iZ) to polysaccharidetest, x 51000.

Fig. 10. After splitting. Persistence of a thin non-reactive extramural coat, x 51000.

13Figs, I I , 12. Spreading of intercellular channel in mung bean. Uranyl acetate - leadcitrate staining; no extraction.

Fig. I I . Note the asymmetry of the channel (s): one side shows a splitting layer (si),the other side is devoid of such a layer (arrows), x 45000.

Fig. 12. Detail of a lateral opening (arrow) in the splitting layer (si), x 62000.Fig. 13. Splitting layer (si) and fusion figure between intercellular channels (s, arrow)simultaneously present. Pea; PATAg staining + DMSO. x 42000.Fig. 14. Corner reinforcement (c) of an intracellular space (s). Pea; PATAg staining+ DMSO. x 67000.

Intercellular gas space opening jfjj

cavity surrounded by pectic substances. As the cavity grows, the previous wall layersand middle lamellae of the parent cells break, become gelatinous or dissolve and thecavity becomes the intercellular space. Comparable interpretations have been adoptedby subsequent authors. These interpretations assign a major role for pectic middlelamellae and suppose a destruction of part of the original wall for the establishment ofthe continuity of the channel.

Figs. 15-17. Ultimate stage; fusion of intercellular channels. Pea. PATAg staining;no extraction.

Fig. 15. Union of channels (s). The residual wall is visible (arrow), x 11000.Fig. 16. Shift of wall (arrows) between 2 channels (s). x 20000.Fig. 17. Detail of a residual wall (arrow), x 20000.

Cytochemical observations at the ultrastructural level show, both in root and inhypocotyl, that the growth of the intercellular spaces is sequential. The progression isbasipetal in the root and basifugal in the hypocotyl. Contrary to current views, walldestruction occurs only as the terminal phase of the programme of wall modificationand is not necessary for the initiation of the aerating system. Likewise, the pecticmiddle lamella does not seem to be directly engaged in the main steps of the process,which appears subordinate to the presence of specialized substructures within the wall.

The following successive stages of wall re-modelling can be distinguished:(1) Differentiation of the splitting layer at the cell junctions. This slender wall layer is

well characterized in its ultrastructure and cytochemical reactivity. Although ratherdifficult to visualize in intact tissue with routine staining, the splitting layer is clearlyrevealed after mild extraction of the tissue. It has a distinctive enzymic activity asindicated by the Gomori test when applied to cryosections. The presence of phos-phatase activity has been reported several times, both by cytochemistry and bio-chemistry in young tissues, especially in root apices (Hall, 1969, 1971; Poux, 1970;Sexton, Cronshaw & Hall, 1971; Hall & Davies, 1971). However, with conventionalincubation of tissue pieces in the medium a number of problems are encounteredconcerning the penetration of the substrate through the bulk of the tissue (Sexton

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334 J- C. Roland

et al. 1971) and the results in the wall are positive but diffuse. The advantages ofultracryotomy and direct incubation of sections is that an equal and direct contact ofcell components with the substrate is achieved. It leads to a more precise localizationof the deposits which in the present case appear specific for the splitting layer.

The deposition of this layer occurs early in development and is achieved at thevery beginning of cell growth. The layer extends unequally from cell junctions tocell facets.

(2) Cleavage of the splitting layer. The pattern of splitting and the superimposedphosphatase activity indicate that the moving apart of the adjacent walls results froma precise autolytic process in the median layer. Typically, the cleavage is achievedgradually from the extremity of the layer. The starting points being lateral, this couldexplain why (if the layer is not visualized or the magnification too low) the firstintramural chinks could appear to arise independently of the preceding intercellularsystem.

(3) Opening of gas channels. The median cleavage of the wall at the cell junctionsallows the moving apart of the adjacent middle lamellae and primary walls. The cellsround off and the intercellular channels become largely opened. They are limited bythe corner reinforcements which appear as the limiting points of extension. Com-parison with fresh tissue indicates that the channels are filled with a continuous gasphase. The predominance of carbon dioxide in this gas phase seems to indicate(Burstrom, 1959) that the channels, running through immature and heterotrophiccells, function primarily as an export system.

(4) Possible wall collapse and final extension of the aerating system. This is a local walltearing and destructive process. Unlike the previous stages which occur in a preciseand characteristic manner, this step occurs in a quite irregular fashion. No specialsubstructure appears to be related to the disappearance of the wall, and it seems thatmerely a destruction of portions of the wall occurs.

The problem of the nature of the surface lining the intercellular space in planttissues is of importance from the physiological viewpoint. It has received muchattention but is still controversial. Microchemical observations by light microscopy,wettability experiments and diffusion resistance suggest strongly that the internalspace is lined with a lipophilic film, possibly comparable to cuticular or suberin layers(see Haiisermann, 1944, and reviews of Scott, 1949, 1966; Martin & Juniper, 1970;and also Jarvis & Slatyer, 1970; Schonherr & Bukovac, 1972; Hayward, 1974). How-ever, the concept of internal suberization is not well documented at the ultrastructurallevel and is not generally accepted. Further studies will be necessary to establish apossible analogy between the extramural coat and cuticle layers. Nevertheless, we canassert that the mechanism of intercellular space development ensures that the gascavity is lined, as soon as it opens, with a continuous non-polysaccharide extramuralcoat and not with hydrophilic, dispersed layer. From the beginning, the coat con-stitutes a thin barrier to diffusion between the aqueous phase (wall medium) and thegaseous phase (intercellular space content).

Various processes in plants involve wall fission, for example, foliar abscission, rootcap cell maturation, callus and in vitro cell production. In these cases alterations of

Intercellular gas space opening 335

the wall have been extensively studied. In the abscission zone (see Addicott & Wiatr,1977; Sexton, 1976, and bibliography therein) the process of wall fission is correlatedwith the appearance of strong pectinase activity which produces a swelling of themiddle lamella. Subsequently a dispersion of the central region of the wall occurs andthe microfibrillar material fills the swollen region between cells. Likewise during rootcap and rhizoplane formation, as well as during callus formation (see Leppard, Colvin,Rose & Martin, 1971; Leppard, 1974; Roland & Pilet, 1972), the wall undergoesmarked swelling of its pectic middle lamella, which progressively changes into adisperse, hydrophilic mucilage which forms an ' intercellular glue' between cells. Inopposition, during the opening of air-space no general swelling occurs and the lyticactivity appears limited to a non-pectic wall structure. One of the remarkable peculi-arities of the opening is that the mechanism involved never leaves the polysaccharidesof the wall naked.

In conclusion, the formation of the aerating system studied here involves a pro-grammed sequence of wall modification in which special substructures are engaged.This is a further example which contradicts the view that the wall is an inert cellcomponent and that changes occurring within it are essentially passive.

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