New Phvtol. (1996), 132. 603-615

Development of water-conducting cells inthe antipodal liverwort Symphyogynabrasiliensis (Metzgeriales)

BY ROBERTO LIGRONE' AND J E E F R E Y G. D U C K E T T ^ *

^ Facolta di Scienze Ambientali, Secondo Ateneo Napoletano, via Arena 22,81100 Caserta, Italy^School of Biological Sciences, Queen Mary and Westfield College, Mile End Road,London El 4NS, UK

{Received 22 May 1995; aeeepted 18 December 1995)

SUMMARY

The thallus of the metzgerialean liverwort Symphyogyna brasiliensis Nees contains a strand of dead thick-walledcells with helicoidally-arranged pits that are presumably involved in water transport. During the first phase ofdifferentiation these cells undergo a 13-16-fold elongation while remaining thin-walled and almost unchanged indiameter. During suhsequent maturation the walls hecome strongly thickened hy deposition of highly electron-opaque material on extraplasmodesmal areas and of transparent material forming collars around plasmodesmata.Whilst the growing wall shows an ordered microfihrillar texture and is strongly reactive to PATAg staining forcarbohydrates, the material associated with plasmodesmata is amorphous and PATAg-negative. A dense corticalarray of microtohules (MTs) overlies the growing wall except in proximity to plasmodesmata, which are closelyassociated with tuhular endoplasmic reticulum (ER). During cellular maturation plasmodesmata undergoextensive secondary elongation hy incorporation of cortical ER supposedly continuous with desmotuhules.Quantitative analysis of plasmodesmal frequencies in relation to cellular elongation and wall thickness indicatesthat there is no de novo formation of plasmodesmata. Cortical MTs, wall microfibrils and secondarily-modifiedpiasmodesmata are consistently co-aligned, all forming helices of about 45°. During maturation the Golgiapparatus proliferates and a vast number of vesicles containing PATAg-positive material are produced from amembrane domain interpreted as trans Golgi network, whilst PATAg-negative vesicles are formed along thefenestrated margins of cis and medial dictyosomal cisternae. Exocytosis of PATAg-positive vesicles is confined toextraplasmodesmal areas. In ageing cells abundant fibrillar material, also positive to PATAg-test, accumulateswithin pleomorphic membrane-bounded tubules. Final cytoplasmic dissolution involves the lysis of all cellularmembranes and the liberation of the membrane-bounded fihrillar material, that is subsequently deposited onto thewalls. The eventual dissolution of the plugs of amorphous electron-transport material results in the formation ofopen pits. Similarities in the cytological mechanisms underlying pore development in water-conducting cells ofSymphyogvna and in the sieve elements of angiosperms are discussed.

Key words: Liverworts, cellular morphogenesis and cortical microtuhules, Golgi bodies, plasmodesmata, water-conducting cells.

vascular plants, the hydroids lack a protoplasmicINTRODUCTION conteiit at maturity and undergo a partial lysis of cell

Specialized water-conducting cells are present both walls during final stages of differentiation. Accordingin mosses and liverworts. Endohydric mosses (Proc- to Hebant (1977), removal of non-cellulosic poly-tor, 1982) possess strongly elongate cells, referred to saccharides from the deeply inclined terminal wallsas hydroids, that form a central strand of variable produces a loose cellulosic network with low re-size in the gametophyte leafy stem and/or sporo- sistance to water flow.phyte seta and in numerous taxa also occur in the Whilst bydroids are widespread in bryoid mosses,leaflet nerve (Hebant, 1977). Like tracheids in i.e. the largest group of extant mosses (cf.

Sphagnales, Andreaeales, Takakiales), specialized

* To whom correspondence should be addressed. water-conducting cells are restricted to a few small

604 R. Ligrone and J. G. Duckett

Table 1. Changes in plasmodesmal frequency and wall thickness during differentiation of water-conducting cellsin Symphyogyna brasiliensis

Stage

Young dividing cellsThin-walled elongating cellsMature living cells

The numbers are means+ SD from at least 20 measurements.* Number of plasmodesmata per 10 ftm wall profile.

Cellularlength{ftm)

13-3 + 2-6180-220260 + 27

Cellularwidth(/im)

8-1 ±2-08-6 ±1-88-6-1-2-2

Plasmodesmalfrequency*

37'0 + 8'l2 '2+M9-2+ 3-0

Wall thickness(/tm)

0-27 ±0-050-28 ±0-111-30^-0-40

Plasmodesmallength(/im)

0-27+0-050-28 + 0-111-42 + 0-36

taxa in liverworts, i.e, the Calobryales and membersof the metzgerialean suborder PallaviciniineaeSchust. As in mosses, water-conducting cells inliverworts lack cyotoplasmic contents at tnaturity.However, in liverworts they occur only in thegametophyte. Moreover, apart from Moerckiahibernica (Hook.) Gott., w^here water-conductingcells have bydrolysed walls apparently similar tothose in moss hydroids (Hebant, 1979), the walls ofwater-conducting cells in liverw-orts are perforatedby distinctive pits. The report by Hebant (1979)of perforations in water-conducting elements inTakakia requires further substantiation because tbisgenus has recently been transferred from liverwortsto mosses (Ligrone, Duckett & Renzaglia, 1993;Smith & Davison, 1993).

Pits are concentrated on terminal walls in theCalobr>-ales (Burr, Butterfield & Hebant, 1974;Hebant, 1977, 1979) whereas they are more scatteredin large numbers on botb longitudinal and terminalwalls in tbe metzgerialean genera Symphyogyna,Pallavicinia and Hymenophyton (Smith, 1966;Hebant, 1977, 1979). Water-conducting cells inCalobryales, though lacking cytoplasmic contents atmaturity, are similar in shape to nortnal parenchymacells. By contrast, water-conducting cells inmetzgerialean liverworts have a distinctive tracheid-like appearance due to their elongate and taperedshape combined with thickened longitudinal wallsand belicoidally arranged pits (Smith, 1966; Grubb,1970; Burr et al., 1974; Hebant, 1977). Experimentalevidence that water moves in these cells at much afaster rate than in neighbouring cortical cells hasbeen obtained by Smith (1966) using eosin as atracer.

Single pits range from 125 to 250 nm in diameterand appear to be ontogenetically related to plasmo-desmata (Smith, 1966; Hebant, 1978, 1980). Thepresence of plasmodesma-derived perforations hasimportant evolutionary implications as this sets tbewater-conducting cells in liverworts clearly apartfrom moss hydroids, which do not have pitted walls,and from tracheary elements in vascular plants,where local lysis of walls is not associated withplasmodesmata but depends on pre-pattemed wall

modifications (Hepler, 1981; Seagull & Falconer,1991).

In spite of their unique cytology, water-conducting cells in metzgerialean liverworts havenever been investigated in detail following thepioneering study by Smith (1966); in particular,very little is known of tbe cytologica! mechanismsunderlying pit development (Hebant, 1978, 1980).Further investigation of these cells is now desirableparticularly in relation to increased interest inproblems pertaining to plasmodesma! structure,development and modifications during cellular mor-phogenesis (for review see Lucas, Ding & Van derShoot, 1993). This paper reports a developmentalstudy of water-conducting cells in a species of thegenus Symphyogyna, with emphasis on plasmo-desmal distribution and modifications from veryearly to mature stages of differentiation.

MATERIALS AND METHODS

Plants of Symphyogyna brasiliensis Nees were col-lected in the Sehlebethebe National Park (Lesotho).Whole thalli, c. 2 cm long, were fixed with a mixtureof 3' 'n glutaraldehyde, 1' o formaldehyde (freshlyprepared from paraformaldehyde) and 0-5 "o tannicacid in 005 M sodium phosphate buffer, pH 7-0, for1 h at room temperature. The specimens were thencut into pieces c. 3 trim long and fixed for anadditional 2 h in fresh fixative. After several rinses inO'l M buffer and post-fixation in 1 °o osmiumtetroxide in 0-08 M sodium phosphate buffer, pH 6-8,overnight at 4 °C, the specimens were dehydratedwith etbanol and embedded in Spurr's resin ac-cording to the procedure previously described(Ligrone & Duckett, 1994). Thin sections were cutwith a diamond knife, mounted on uncoated gridsand sequentially stained with 5 "o methanolic uranylacetate for 15min and basic lead citrate forlOmin. The periodic acid/thiocarbobydrazide/silverproteinate (PATAg) test for insoluble carbohydrateswas performed according to Roland & Sandoz (1969)on sections mounted on uncoated gold grids. Eitherperiodic acid oxidation or treatment with thiocarbo-

Water-conducting cells in the liverwort Symphyogyna 605

Figure 1. Water conducting cells in Symphyogyna. {a)-ic), light microscopy, ia), Transverse section of arecently bifurcated thaiius. Two inner strands of narrow cells are visible (arrows), x 190. {b). Highermagnification of a strand at the end of the differentiation process. The intiermost cells are fully mature atidappear empty, whilst more peripheral cells contain densely staining material, x 1050. ic). Aniline-bluefluorescence in the central strand in a fresh section of the mature thallus. x 650. id-e), Electron microscopy.id). Detail of a mature dead cell. Note the thick electron-opaque walls and pits (arrows). The lumen is almostcompletely free of contents, whilst electron-opaque material is still abundant in neighbouring cells, x 5150. {e).Vertical longitudinal section through a thallus apex showing an immediate derivative of the apical cell (above)and initials of the strand (below). Note the high frequency and even distribution of plasmodesmata in the initialcells; nucleus (n). x 6700.

606 R. Ligrone and jf. G. Duckett

Figure 2. Early stages of differentiation of water conducting cells in Symphyogyna. ia). Elongating thin-walledcell; note the large vacuoles (v) and the relatively small nucleus (n). x 4850. {b). Cell at the onset of maturation.The walls are still thin but have become much denser, whilst the nucleus (n) is larger and more irregular inoutline; plastids (p) are relatively large and contain abundant starch, x 5350. (r). Cells at a more advanced stageof maturation; tbe walls are thicker and show numerous developing pits (arrowheads); plastids (p) are morepleomorphic; nucleus (n). x 5600. {d). Photomicrograph showing a longitudinal section of water conducting

Water-conducting cells in the liverwort Symphyogyna 607

hydrazide was omitted for controls. The ultra-structural observations were performed witb a Jeol1200 EX2 or a Philips CM12 electron microscope.

Half-micrometre-tbick sections were stained witb1 % toluidine blue in 1 % borax and photographedunder differential interference contrast optics with aLeitz Ortholux microscope. The same microscope,equipped with epifiuorescence optics and the ap-propriate filters, was used for callose detection inwhole thalli and hand-cut transverse sections stainedwith 0-1 °o water soluble aniline blue in 0-067 MKH.PO^ at pH 8-5 (O'Brien & McCully, 1981).

Changes in cellular sizes during differentiationwere analysed on 1 //m-thick longitudinal sectionsstained with toluidine blue. Cbanges in plasmo-desmal frequency and wall thickness were analysedon 20 randomly selected micrographs ( x 6000 finalmagnification) from non-serial golden sections foreach of the following stages: (1) young dividing cells,(2) elongating thin-walled cells, (3) elongate thlck-walled cells prior to cytoplasmic dissolution. Onlylongitudinal walls were considered for analysis; tbeoverall wall length, analyzed from at least 12 differentcells, was 115 //m for stage (1), 845 //m for stage (2)and 925 //.m for stage (3). Plasmodesmata were scoredonly when a desmotubule profile was visible. Wallsleeves lacking a plasmodesmal profile were notscored, w^bilst branched plasmodesmata were scoredtwice.

A diminution of plasmodesmal frequency is to beexpected as a consequence of cellular elongation.provided tbat no de novo plasmodesmal formationoccurs. On the other hand, the probability that aplasmodesma, however orientated, falls within asection (and is therefore scored) is directly pro-portional to the plasmodesmal length. Thus, com-parison of plasmodesma] frequency at tbe threedevelopmental stages considered in this study needsthe values to be normalized in relation to changes incellular and plasmodesmal length.

Normalization bas been performed according totbe follow ing relation :

where /;, is the normalized frequency, F the meanfrequency actually measured, Cl^ and P/,. tbe ratiobetween cellular and plasmodesmal length at thestage 2 or 3 and the corresponding figure at stage 1.On the basis of the data reported in Table 1, Cl^ andPl^ are c. 20 and 5 at the stage 3 (considering only themean values of cellular and plasmodesmal lengtb).

whilst during tbe elongation phase (stage 2) Cranges between 13-5 and 16-5, and Pl^ is about 1.

R E S L' L T S

Transverse sections of the thallus show one or, justbehind dichotomies, two strands of 25-60 narrowcells (Fig. 1 a). At maturity' these cells lack cyto-plasnnic contents and bave thick, densely stainingwalls with numerous pits of varying sizes (Fig. 16,d). In sections from the subapical region of thethallus the walls of inner cells show a bright yellowfluorescence after staining with water-soluble anilineblue (Fig. 1 c). No autofluorescence was observed inunstained material nor in material stained withaniline blue at low pH.

Origin and elongation of strand cells

The strand arises from subapical meristematic cellsproduced by the activity of the single apical cell(Renzaglia, 1982) (Fig. \e). Differentiation beginsafter a few divisions and is virtually completedc. 0'7-0-8 mm bebind tbe apical cell. A quantitati\ eanalysis of morphological changes during differen-tiation is set out in Table 1.

Young cells at the beginning of differentiation areshortly rectangular and show a high frequency ofplasmodesmata on both longitudinal and transversewalls. During differentiation the cells elongate about20-fold while remaining almost unaltered in di-ameter (Table 1) and their terminal walls becomedeeply inclined. Cellular elongation is associatedwith a sharp increase in plasmodesmal frequency(Table 1). The initial increase in cellular volume ismostly due to enlargement of the vacuoles (Fig. 2a).Tbe cell walls, underlain by a dense array of corticalmicrotubules, remain \'ery thin and relativelyelectron-transparent until length bas increased13-16-foId (Fig. 2e). At tbis stage a sudden increasein the electron-opacity of the wall (Fig. 2b, f) signalstbe onset of the maturation process leading to thedevelopment of the thick and highly perforated wallstypical of mature strand cells.

Maturation of strand cells

Cytoplasmic changes. With the onset of maturationtbe strand cells sbow cytoplasmic proliferation whilstthe vacuoles become snnaller and more numerous (cf.Figs 2b, c, 2b). The nucleus enlarges and becomes

cells (arrows) at the same stage as in (c) and adjacent parench\Tna cells, x 380. (e-h). Details of the wal! duringdifferentiation, (e). Elongating cell; note the numerous cortical microtubules (arrowheads) and plasmodesmata(arrows), x 55 000. (/), Cell at the onset of maturation; plasmodesmata (arrows), x 53 500. (g), Thickening wall;note the developing collars of electron-transparent amorphous material and endoplasmic reticulum(arrowheads) associated with the plasmodesmata. x 38 500. (h). Profile of smooth endoplasmic reticulum(arrowheads) associated with a plasmodesma and continuous with more internal rough endoplasmic reticulum(er). X 45 500,

608 R. Ligrone and y. G. Duckett

Figure 3. Details of water conducting cells in Symphyogyna at advanced stages of maturation, (a). Glancingsection of cortical cytoplasm, showing irregularly branched smooth endoplasmic reticulum (asterisks) andcortical microtubules (arrowheads) co-aligned with microfibrils in the underlying wall, x 38 500 (b and c). Cellsjust before and immediately after cytoplasmic dissolution. Note the abundant electron-opaque deposits in thelumina (arrows), x 4900. (d). Detail of the wall in a cell at the same stage as in (6), showing developing pitsassociated with plasmodesmata; the arrows point to two plasmodesmata merged into a single channel on both

Water-conducting cells in the liverwort Symphyogyna 609

irregularly elongate in shape (Fig. 2a—c). Theplastids lose their starch content, becomeincreasingly pleon^orphic and ultimately form longand thin sheets encasing portions of cytoplasm (cf.Figs 2b, c, 4g).

The cytoplasm of maturing cells is rich indictyosomes and associated vesicles. These are oftwo clearly distinct types: (1) vesicles containingfibrillar material of medium electron-opacity; thesevary in size, arise from the fenestrated margins Golgicisternae (Fig. 46) and are negative to PATAgstaining (Fig. 4-d). (2) vesicles with extremelyelectron-opaque contents; these are much moreabundant, have a relatively constant diameter ofc. 110 nm, arise from part of a tubular membranousnetwork associated with the trans side ofdictyosomes, and are strongly positive to the PATAgtest (Fig. 4d). Coated vesicles were observed rarely.The electron-opaque material in PATAg-positivevesicles appears to be exported into the periplasmicspace by exocytosis (Fig. 5d) and is then incor-porated into the wall (Fig. 4e). Such presumedexocytosis of PATAg-positive vesicles is restrictedto plasmalemmal areas away from the pits. In almostmature cells the membrane network with PATAg-positive vesicles is much less m evidence andelectron-opaque material accumulates in membrane-bounded pleomorphic tubules (Figs 3 b, 4/) whilstPATAg-positive vesicles become less abundant.This material exhibits similar staining properties tothe material in PATAg-positive vesicles, includingstrong reactivity to the PATAg test (not shown), butdifferent from these it reveals a clearly fibrillarstructure at high magnifications (Fig. 4/) .

Wall modifications. Wall maturation involves thedeposition of extremely electron-opaque material onextraplasmodesmal areas (i.e. the wall proper) and ofnearly electron-transparent amorphous materialforming distinctive collars, about 230 nm±50 SD indiameter, around single plasmodesmata (Fig. 2c, g).The piasmodesmata and associated collars are evenlydistributed in both transverse and longitudinal walls.Cellular elongation ceases shortly after the beginningof wall maturation. At this stage the cells are highlyelongate and tapered at both ends (Fig. 2d).

The v 'all of maturing strand cells is heavily stainedby toluidine blue as well as by the standard stainingprocedure for electron microscopy; moreover it isstrongly reactive to the PATAg test (Fig. Sb). Bycontrast, the material associated with plasmodesmata

is weakly stained with the standard procedure and isnegative to PATAg (Fig. Sb).

Modifications of plasmodesmata. In the course ofstrand cell maturation the plasmodesmata elongateto about five times their original length, thusaccommodating the increasing thickness of walls.During this process stnooth-surfaced elements ofendoplasmic reticulum (FR) are regularly found inassociation with both ends of desmotubules (Figs 2g,Ae). These elements are part of a highly anasto-mosing cortical endomembrane system (Fig. Za)showing local continuities with rough ER deeperwithin the cytoplasm (Fig. 2h). Plasmodesmalelongation appears to involve incorporation of thetubular ER elements that are associated and pre-sumably in continuity with desmotubules. Ulti-mately the plasmodesmata exhibit a sinuous shape(Fig. 3(/), with only their mid portion derived fromthe primary plasmodesmata present in young cells.During maturation, the primary wall area surround-ing the mid portion of plasmodesmata becomeselectron-transparent and consequently the collars oneach side become continuous (Figs 3(/, Ae).

The quantitative analysis of cells at an advancedstage of maturation reveals a remarkable increase inthe frequency of plasmodesmata (Table 1). Offrequent occurrence in these cells are branchedplasmodesmata (Fig. 'id) almost certainly formed byencasement by the electron-transparent wall materialof branched ER tubules initially associated with twoadjacent primary desmotubules. Once encased incylinders of transparent wall material, the ERtubules constrict to a minimal diameter, formingtypical desmotubules (Fig. 3e). The space betweenplasmalemma and desmotubule is filled withelectron-opaque material obscuring the boundariesbetween these two components (Fig. 3e). Primaryand secondary regions of plasmodesmata show nostructural differences and have a constant diameterof about 32 nm. Profiles of developing plasmo-desmata with ER that has not yet collapsed areoccasionally visible in glancing sections through thewall-cytoplasm interface (Fig. 3/). In a few casesmembranous tubules were observed within anenlarged periplasmic space between the cell wall andplasmalemma (Fig. 3^). At higher magnification(Fig. 3/7) these tubules appear to comprise twotightly appressed membranes, with highly electron-opaque material associated with the luminal side ofthe inner membrane (Fig. 3^). The tubules range in

sides of the wall during wall growth, x 15000. (c). Transverse section through the mid-region of aplasmodesma; the sleeve between the plasmalemma and desmotubule is filled with electron-opaque material.X 150000. (/), Transverse section of a plasmodesma near the cytoplasm, showing non-appressed endoplasmicreticulum (arrowhead) outlined by the plasmalemma. x 150000, (§), Membranous tubules (arrows) in theperiplasmic space, x 22000, (//), Detail of a membranous tubule showing an outer and inner membrane, thelatter associated with electron-opaque material (arrowheads), x 120000, (/), Membranous tuhule with areduced lumen almost occluded with dense material; microtubules (arrowheads) x 72 500.

610 R. Ligrone and y. G. Duckett

Figure 4. Details of maturing water-conducting ceils in Symphyogyna. {a). Proliferation of the Golgi apparatusand production of abundant vesicles; dictyosome (d). x 27000. {b). Vesicles with fibrillar contents (arrowheads)arising from the fenestrated margin of a dictyosomal cisterna. x 58 500. (f). Vesicles vtith electron-opaquecontents associated with pleomorphic membranous tubules, x 51 000. {d), PATAg staining; the vesicles witha dense core are strongly reactive (arrowheads) whilst the others are not (arrows), x 48000. {e). Dense material.

Water-conducting cells in the liverwort Symphyogyna 611

diameter from r. 110 to 50 nm, with the densematerial almost completely filling the lumen of themore attenuated regions (Fig. 3?).

Maturing cells exhibit a well-developed corticalarray of microtubules (MTs) parallel to microfibrilsin the adjacent wall, both systems forming helices ofabout 45° relative to tbe long axes of the cells (Fig.3 a). Though MTs are mucb scarcer in the corticalcytoplasm underlying plasmodesmata and theassociated sleeves of electron-transparent wall ma-terial, tbese follow the same helicoidal pattern as theMTs and microfibrils in the adjacent electron-opaque wall areas (Fig. 5a). Cells in the samelongitudinal file exhibit helicoidal patterns with thesame orientation, whilst cells of adjacent files bavethe opposite orientation (Fig, 5a). It becomesevident, in final stages of differentiation, that thehelicoidal arrangement of the plasmodesmata andassociated electron-transparent material is onto-genetically related to the distinctive helicoidal pat-tern of pits m mature cells.

Along the boundary between the central strandand cortical parenchyma, the wall and plasmodesmaldomains belonging to cortical cells are not affectedby the maturation process operating in tbe adjoiningwater-conducting cells (Fig. 5 c). At advanced stagesin development plasmodesmata on the cortical cellside are obliterated by deposition of new wall layers(Fig. 5d).

Cvtoplasmic dissolution and the formation of pits.Final protoplasmic degeneration in mature cellsappears to be engendered by disruption of theplasmalemma, tonoplast and other cytoplasmicmembranes as well as of the plasmodesmata (Figs 3r,5e).

Dissolution of the material forming the collararound plasmodesmata in conducting cells startswitb cytoplasmic degeneration (Fig. Se) and iscomplete after tbe cellular iumen has become free ofthe fibrillar material and otber cytoplasmicremnants. This produces incomplete pits in wallsabutting cortical cells (Fig, 5/), whilst complete pitsare formed in the internal v^alls (Figs It/, 5^). Thediameter of pits is often reduced in the middle due toincomplete dissolution of the primary wall septum(Figs \d, 5g). Fusion of neighbouring collars is offrequent occurrence during development (Fig. 5 a)and results in tbe formation of pits wider tban asingle sleeve (Figs \d, 5g). Following n:iembranedisruption and cellular death, tbe fibrillar material,now free in the cellular lumen, appears to beincorporated into the walls (Fig. 5e) whilst othercytoplasmic remnants disappear rapidly, probably

because of the activity of lytic enzymes. Conse-quently water-conducting cells are completely de-void of contents at maturity (Fig. 1 b).

D1 s c I j s I o N

The development of water-conducting cells inSymphyogyna involves an ordered sequence ofevents, including differential wall thickening, cyto-plasmic autolysis and localized wall dissolution, thatis quite similar in complexity to tbe ontogeny oftracheary and sieve elements in vascular plants.

Cortical MTs and the helicoidal patterning of pits

Following elongation, during which the wall thick-ness does not change appreciably, tbe cells enter amaturation phase characterized by a four- to six-foldincrease in wall thickness. Whilst tbe wall isthickened by deposition of dense material combinedwith spirally-arranged microfibrils, electron-trans-parent collars of amorphous material are depositedaround plasmodesmata. The heterogeneous patternof wall thickening is mirrored by the arrangement ofcortical MTs; these are abundant beneath tbegrowing wall except in the regions facing developingpits. Cortical MTs exhibit a similar non-uniformpattern in differentiating tracbeary elements, wherethey appear to be involved in localized deposition ofcellulose and perhaps of other wail componentsduring tbe development of secondary wallthickenings (Hepler, 1981; Schneider & Herth,1986; Seagull & Falconer, 1991; Hogetsu, 1991).Likewise, cortical MTs in differentiating sieveelements of wheat overlie the lateral walls inabundance except near plasmodesmata and areentirely absent from tbe end walls, which e\ entuallydifferentiate into sieve plates (Fleftheriou, 1990,1991).

Tbe helicoidal pattern of pits in water-conductingcells of metzgerialean liverworts was interpreted bySmith (1966) as the result of stretching due tocellular elongation. Tbis hypothesis, bowever, doesnot explain the different orientation of pits inadjoining cells, also noticed by Smith (1966) inSymphyogyna circinata Nees & Mont. Moreover,wall growth and pit development in water-con-ducting cells of 5. brasiliensis take place for tbe mostpart after elongation bas terminated. Our obser-\ations suggest that the spiral arrangement ofplasmodesmata and associated pits is generatedmechanically by the spiral microfibrillar texture ofthe growing wall and perhaps ultimately depends,according to current models (Giddings & Staehelin.1991), on tbe pattern of the cortical array of MTs,

liberated by exocytosis, is being incorporated into the fibrillar texture of the wali (arrowheads); note theplasmodesma associated with a collar of electron-transparent material and ER tubules at both ends (arrows),X 36 500. (/), Deposit of fibriilar material within a membranous tubule in a cell approaching maturity. X 41000.

(g). Highly pleomorphic plastids (arrows) in a maturing cell, x 36000.

612 R. Ligrone and jf. G. Duckett

Figure 5. Details of the wall of maturing water-conducting cells in Symphyogyna. (a), Longitudinal sectionshowing co-alignment of cortical microtubules (arrows), wall microfibrils and developing pits; note theopposite orientation of the helicoidal pattern in the two adjacent cells. Arrowheads point to the fusion ofdeveloping pits, x 1800. (b), PATAg staining; the wall is strongly positive whilst the material in developing pits(arrows) is negative, x 24000. (f). Interface between the inner strand (left) and cortical parenchyma (right);developing pit contiguous with a normal plasmodesma (arrowhead) in adjoining parenchyma cell, x 26500. (d),Same area as in (<-), at a more advanced stage; the plasmodesmata on the parenchyma cell side have beenobliterated by deposition of new wall layers (arrows), x 38000. (t). Plugs persisting in the pits in a recentlydifferentiated cell; arrowheads pomt to dense fibrillar material being deposited onto the wall, x 32000, (,/"),PATAg staining; dissolution of the amorphous electron-transparent material has resulted in the formation ofpartial pits (arrowheads) in the common wall between a conducting cell (left) and a cortical parenchyma cell(right). Note the irregular outline and relatively loose texture of the inner part of the conducting cell wall(arrowed), x 22000, (g). An open pit between fully mature conducting cells; remnants of the primary wall arevisible in the mid-region (arrowheads), x 19 500.

Water-conducting cells in the liverwort Symphyogyna 613

Wall heterogeneity and the origin of the pits

Differences in morphology and staining propertiesindicate clearly that the wall proper andplasmodema-associated material have a differentcon-iposition. On the basis of histochemical andextraction tests. Smith (1966) concluded that thewalls of water-conducting cells in Symphyogynacircinata mainly consist of cellulose and hemi-cellulose whilst pectic substances and lignin arelacking, ln contrast to the negative reports by Smith(1966) and Hebant (1978). aniline blue inducedbright fluorescence in the strand cells in freshmaterial of S. brasiliensis, thus indicating the pres-ence of callose. However, this test did not permitdiscrimination between the wall and de\"eiopingpits.Identification of the material deposited arounddeveloping pits as callose is consistent with itsamorphous electron-transparent appearance and lackof reactivity to PATAg staining (Roland, 1978). Thisis in line with the notion that liverworts are able toproduce callose not only in the cells of the sporo-genous lineage (Brown & Lemmon, 1987) but alsoin somatic cells (Lerchl et al., 1989). Our observa-tions, howe\'er, are not conclusive since aniline blue-induced fluorescence was observed in young as wellas mature parts of the thallus whilst the amorphousmaterial is remo\ed from pits during final matu-ration. Thus the possibility that fluorescence isartefactual in origin (O'Brien & McCully, 1981)cannot be excluded.

Whatever the nature of the amorphous material,the cytological mechanism underlying pit devel-opment in water-conducting cells of Symphyogyna isstrongly reminiscent of the mechanism of poredevelopment in sieve elements of angiosperms(Evert, 1977, 1990; Esau & Thorsh, 1984, 1985;Lucas et al., 1993). In both cellular types theformation of pores involves differential wall thick-ening with deposition of special collars aroundplasmodesmata, elongation of plasmodesmata byencasement of cortical ER elements presumptivelycontinuous with desmotubules, localized dissolutionof primary wall and final remo\'al of tbe collars andplasmodesmata. The collar deposited around plasmo-desmata in sie\e elements consists of electron-transparent material thought to be callose (Evert1977, 1990; Esau & Thorsh 1984. 1985; Lucas etal.1993), whilst the material in developing pitsof Symphyogyna, though lacking any visibleorganization, is not completely transparent toelectrons. The same has been observed in othermetzgerialean liverworts (Smith 1966; Hebant1978). Chlortetracycline-induced callose depositionin thallus cells of the liverwort Rie/la helicophylla isconsidered by Lerchl et al. (1989) to involve calloseaccumulation within vesicles presumptively arisingfrom partially-coated reticulum, followed by exo-cytosis. The uitrastructural evidence obtained in the

present study does not support an exocytotic path-way for plug formation in Symphyogyna. Here, assuggested for callose deposition in sieve elements(see Evert, 1990 for review), the amorphous materialis more likely to be syntbesized at the level of theplasmalemma, possibly from precursors transportedvia ER. According to this model, whilst MT-associated plasmalemma regions are involved in thesynthesis of orderly textured cellulose microfibrils,tbe areas associated with ER and plasmodesmatawould produce the amorphous material, possiblycallose-like polysaccharide and/or disorganizedcellulose microfibrils. It bas been suggested thatcellulose synthase and callose synthase, bothlocalized in the plasmalemma, might share commonsubunits wbich are regulated in an opposite fashionin vivo (Deimer, 1987, 1991). The association of ERwith developing pits in water-conducting cells ofSymphyogyna, as in sieve elements of angiosperms(Esau & Tborsh, 1984, 1985), suggests a possibleinvolvement of ER in the control of plasmalemmaglucane synthase activity.

in striking contrast to sieve elements, the removalof plugs from the pits in water-conducting cells ofSymphyogyna is preceded by complete cytoplasmicdegeneration. This is probably an extremely rapidevent, since no intermediate stages could be foundbetween those shown in Eig. 2h, c. Possibly the finaldegeneration is related to obliteration of plasmo-desmal connections with adjacent parenchyma cellsand consequent symplasmic isolation of the strandcells.

Secretory activity and wall maturation

Our observations provide direct evidence for par-ticipation of PATAg-positive vesicles in the buildingof the wall matrix in water-conducting cells. Onceexported outside of the plasmalemma. the PATAg-positive material is incorporated into the wall andprobably represents the main source of non-cellulosic wall polysaccharides. Different from thePATAg-positive vesicles, in\olvement of PATAg-negative vesicles in exocytosis could not be es-tablished in this study. Whilst the latter showcontinuity witb, and therefore probably arise from,cis and medial Golgi cisternae, the membranedomain producing PATAg-positive vesicles is topo-logically identifiable as tbe /raws-GoIgi network(Zhang & Staehelin, 1992). This component of theGolgi apparatus, also know-n in plant cells as thepartially coated reticulum (Pesacreta & Lucas. 1985),is thought to control final sorting and targeting ofsecretory material (Staehelin et al., 1991). Thismembrane domain in Symphyogyna differs in onerespect, howe\er, from typical trans-Golgi network,namely its paucity of coated vesicles.

A recent immunogold study in suspension-cultured sycamore maple cells has revealed that the

614 R. Ligrone and J. G. Duckett

assennbly of xylanoglucan, tbe major type of hemi-cellulose of dicotyledonous cell walls, is confined tothe trans Golgi cisternae and trans-GoXgi network(Zhang & Staeblin, 1992). Combined witb theobservation that hemicellulose is tbe major non-cellulosic polysaccharide in Symphyogyna (Smitb,1966), this finding suggests that the contents ofPATAg-positive vesicles is mainly hemicellulose.The fibrillar material accumulating in ageing cellsalso appears to arise from the trans-Go\g\ network ina final burst of secretory activity culminating withcellular death. Like the contents of PATAg-positi\'evesicles, this material is heavily stained by thePATAg method for carbohydrates and is incor-porated into tbe wall; however, unlike the contentsof exocytotic vesicles, this fibrillar material isdeposited onto the wall after the dissolution of thecytoplasm. At this stage cytoplasmic mechanismscontrolling exocytosis, possibly including corticalMTs and cortical ER (Hepler et al., 1990; Battey &Blackbourn, 1993), are no longer present. Oblit-eration of pits by the fibrillar material is probablyprevented by plugs of amorphous material persistingin the pits for some time after cellular death.

Modifications of plasmodesmata

During cellular elongation preceding wall thickening(stage 2), the normalized plasmodesmal frequency isin tbe range 30^5, i.e. much the same as during thestage 1, Thus the declme in the density of plasmo-desmata observed in longitudinal walls during stage2 is wbolly consistent with a progressive increase inthe spacing of the plasmodesmata initially present inyoung cells. Likewise, the normalized frequency atstage 3 is about 37, a value remarkably close to tbefrequency measured at stage 1. Tbis indicates thatthe increase in plasmodesmal frequency obser\'edduring cellular maturation is but a statistical effect ofplasmodesmal elongation. Therefore, unlike othersystems (Schnepf & Sych, 1983; Seagull, 1993;Steinber & Kollmann, 1994; see also Lucas et al.,1993 for review), water-conducting cells ofSymphyogyna do not form new plasmodesmataduring differentiation,

Tbe mechanism of plasmodesmal elongation byencasement of cortical FR in the thickening wall asobserved in Symphyogyna is remarkably similar tothe mechanism described for de novo plasmodesmalformation in heterografts (Koilmann & Glockmann,1991) and in regenerating Solanum protoplasts(Monzer, 1991); most likely the same basic mech-anism is involved in the formation of secondaryplasmodesmata in a variety of plant tissues (Lucas etal., 1993). Whilst the term primary plasmodesmatarefers unequivocally to those plasmodesmatadeveloping with the nascent wall during cell division,tbe term secondary plasmodesmata is currently usedboth for de novo-iovmad plasmodesmata and for

secondarily modified plasmodesmata of primaryorigin (Robards & Lucas, 1990; Kollmann &Glockmann, 1991; Monzer, 1991; Lucas et ah,J993; Wolf & Lucas, 1994). According to ourobservations the plasmodesmata present in water-conducting cells in Symphyogyna are an example ofthe second type of secondary plasmodesmata.

Secondary plasmodesmata in Symphyogyna aresimilar in structure to plasmodesmata in Azolla roots(Overall, Wolfe & Gunning, 1982), wath no en-largement in the cytoplasmic sleeve nor in theappressed ER or desmotubule (see Lucas et al., 1993for terminology). We tentatively interpret the mem-branous tubules observed in tbe periplasmic space asER tubules entrapped in localized accumulations ofelectron-transparent wall material. These tubulesare tigbtly ensheathed by the plasmalemma but areneither connected to plasmodesmata nor do tbeyform desmotubules, though the wbole complex may-contract to a diameter not much larger than tbat of anormal plasmodesma.

A C K N O W L E D G ti M 1£ N T S

A British Council-sponsored LINK between Queen Mary& Westfield College and the National University ofLesotho enabled the authors to collect Symphyngyna fromLesotho. This study was in part supported by CNR as apart of a Bilateral Research Project Italy/United King-dom. R,L. thanks Queen Mary Si Westfield College forlaboratory facilities during his sabbatical leave in 1492-3.

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