Microtubules and cell wall development in differentiating protophloem

sieve elements of Triticum aestivum L.

E. P. ELEFTHERIOU

Department of Botany, University ofThessalomki, GR 54006 Thessaloniki, Greece

Summary

The densities of microtubules (MTs) along thelateral walls of developing sieve elements in rootprotophloem of wheat have been investigated byelectron microscopy. They increase gradually inthe very young sieve elements to reach a maxi-mum just before the initiation of wall thickening.During wall increment MTs remain at high den-sities (more than lOMTs^m"1), but their numberdeclines abruptly when wall material depositionceases.

Cell wall thickening is not uniform: broadridges alternate with narrow depressions, thelatter occupied by plasmodesmata. During wallmaterial deposition MTs overlie the thickeningsonly, being entirely absent from the non-thick-ened areas. The orientation of MTs reflects that ofthe currently deposited cellulose microfibrils in

the cell wall, all being perpendicular to the direc-tion of cell expansion. Numerous vesicles, appar-ently of Golgi apparatus origin, are encounteredamongst the cortical arrays of MTs. Though theleast spacing between the contiguous MTs ismuch smaller than the diameter of even thesmallest vesicles, the latter were seen amongstthe MTs, indicating that MTs do not prevent thevesicles from passing between them towards thedeveloping area.

All results favour the suggestion that MTs insieve elements are involved in cell wall patterndevelopment, cellulose microfibril orientation,and presumably in cell elongation.

Key words: wheat, protophloem sieve elements,microtubules, cell wall thickenings.

Introduction

Cell shape in plants is determined by the cell wall andits interaction with turgor pressure (Green, 1980).Explaining the mechanism of cell wall formation and inparticular how the pattern and orientation of thereinforcing cellulose microfibrils arise is therefore es-sential for the understanding of cytomorphogenesis.The process by which green plants produce cellulosemicrofibrils and organize them into a functional wall islargely unknown. Because many studies have impli-cated the plasma membrane as the site of cellulosesynthesis (see e.g. Giddings et al. 1980; Doohan &Palevitz, 1980; Sjolund & Shih, 1983) the relationshipbetween plasma membrane and any cytoplasmic el-ement that might be involved in cellulose apposition isof great significance. Microtubules (MTs), since theirdiscovery in plant cells (Ledbetter & Porter, 1963),have been the most important cellular element stronglyimplicated in defining the pattern and orientation ofcellulose microfibrils in primary and secondary wall

Journal of Cell Science 87, 595-607 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

formation by a substantial body of observations (seee.g. Pickett-Heaps, 1967, 1974; Hepler&Fosket, 1971;Maitra & De, 1971; Doohan & Palevitz, 1980).

Tracheary elements and stomatal guard cells havereceived considerable attention in studies of cytomor-phogenesis for they possess specific wall patternsrelated to cell shape and function (Hepler & Palevitz,1974; Galatis, 1980; Hepler, 1981; Palevitz, 1982). Indifferentiating xylem elements, for instance, MTs haverepeatedly been found to occur next to the cytoplasmicface of plasma membrane lining the bands of growingsecondary wall thickenings and being oriented parallelto the cellulose microfibrils (Wooding & Northcote,1964; Cronshaw & Bouck, 1965; Picket-Heaps, 1967,1974; Newcomb, 1969; Hepler & Fosket, 1971; Maitra&De, 1971).

Unlike tracheary elements, in sieve elements thefeatures of MTs have been investigated to a morelimited extent. Although MTs have been reported tooccur in differentiating sieve elements by many re-searchers (Bouck & Cronshaw, 1965; Tamulevich &

595

Evert, 1966; Behnke & Dorr, 1967; Esau & Gill, 1973;Evert, 1976; Melaragno & Walsh, 1976; Dute & Evert,1977; Esau, 1978; Hardham & Gunning, 1979; Singh,1980; Eleftheriou & Tsekos, 1982; Thorsch & Esau,1982; Fjell, 1986), almost all of them merely mentiontheir presence next to the plasma membrane, and onlyoccasionally refer to their orientation.

The most detailed investigations on MTs in sieveelements have been presented in roots of Azollapinnata (Hardham & Gunning, 1979), Gossypiumhirsutum (Thorsch & Esau, 1982) and Triticumaestivum (Eleftheriou, 1985). The differentiating sieveelements of Azolla continued to elongate and to inter-polate MTs at a high rate. Thorsch & Esau (1982) havecorrelated the appearance and abundance of MTs incotton roots with features of the sieve element proto-plasts. Eleftheriou (1985) studied the MT involvementin ontogenetic (formative) divisions of protophloemsieve elements in roots of wheat. A thorough studytherefore of MT implication in cell wall pattern devel-opment in sieve elements, similar to those carried outfor xylem elements, is missing.

It is the purpose of the present paper, which is acontinuation of and complement to a previous one onthe same species (Eleftheriou, 1985), to consider thepattern of cell wall material apposition in differen-tiating sieve elements of root protophloem of wheat.The abundance, distribution and orientation of MTs,and their relationship with other cellular structureswere examined and correlated with the developmentalstages of the cells in order to find out whether and, ifso, how they are involved in sieve element morphogen-esis.

Materials and methods

Root tips of seminal roots of Triticum aestivum L. cv. MansHuntsman were prepared for electron microscopy as de-scribed (Eleftheriou, 1985).

The differentiating protophloem sieve elements in roots ofwheat are arranged in vertical files (Fig. 1). Each file exhibitsa developmental gradient from young cells near the apextowards more mature regions at the base of the root. Tosimplify the classification, the youngest cells in the files arereferred to as sieve elements despite their lack of distinguish-ing cytological features (cf. Thorsch & Esau, 1982). Theposition of each element within the series is defined by asingle numeral, starting numbering with a readily recogniz-able sieve plate encountered at about 550-580 fim above theroot apex (Fig. 1). This sieve plate lies between the lastimmature differentiating sieve element bearing ribosomes(found at a stage just before the autolysis of many of itscellular components), and the first sieve element deficient instainable material (Fig. 2). The structural difference betweenthe two cells is so sharp that it renders the common sieve platereadily identifiable and capable of providing a reliable start-ing point for sieve element numbering. Numbering proceeds

both downwards and upwards from this point in consecutivenumerals; for distinction, numerals for sieve elements de-ficient in cytoplasm are accented (Figs 1,2). According tothis system the sieve elements are defined by a single number,which readily indicates their position (distance in number ofcells) in relation to the starting point, and sieve plates by twonumerals corresponding to the sieve elements they separate.The sieve plate treated as the starting point for numbering is,for instance, defined as 1-1'.

The present investigation was carried out on longitudinalsections. MTs along the cell walls were counted andexpressed as the number per unit length of cell wall(Hardham & Gunning, 1979). Counts were carried out onelectron micrographs (like those published here), but, inorder to save time and photographic material, and be able totake into account as many samples as possible, many counts(about half) were made directly on the microscope screen athigh magnifications (X 80 000). Considering the diameter ofthe illuminated screen and the image magnification it wasquite simple to determine the wall length under observation.

Results

At the very early stages of development the future sieveelements are ultrastructurally indistinguishable fromthe neighbouring meristematic cells. They are identifi-able as such only by their position within the verticalseries. The youngest cells in the best longitudinalsections obtained were sieve elements (SEs) 16 or 15,that is those found at a distance 16 or 15 cells far fromthe starting point for numbering, namely the sieveplate IT ' . The lateral walls of the very young sieveelements are thin and have a crenulated outline (Figs 3,4). Interphase MTs are spaced out rather evenly alongthe lateral walls, and are found also in close vicinity toplasmodesmata (Fig. 3). A gradual increase in thenumber of cortical MTs is perceptible even in thosevery young sieve elements such as SEs 16-11 (cf.Figs 3, 4).

In the span of SEs 10-7 the lateral walls becomestraightened and uniform, but lack any noticeable wallincrement (Figs 5, 6). At this stage the number of MTsalong the lateral walls increases to a maximum, which isthen maintained (Fig. 17).

The first clear signs of wall material apposition areobserved in SE 6 (cf. Figs 6, 7), which becomegradually more evident in SEs 5, 4, 3, 2 and 1(Figs 7-13). Thickening is not uniform: wall materialis deposited in the areas between the plasmodesmatasites, while the latter areas thicken only very slightly.This pattern of wall development results in a highlyirregular configuration of its inner surface consisting ofbroad thickened areas alternating with narrow non-thickened ones (Fig. 11). The latter are occupied byplasmodesmata that are associated with endoplasmicreticulum (ER) cisternae (Figs 8, 9). The plasmo-desmata show the typical ultrastructural features

596 E. P. Eleftheriou

880-

820-

760-

700-

640-

5 5 8 01

520-

460-

400-

340-

• 6 • ; -

Fig. 1. Light micrograph of two differentiating protophloem sieve tubes in longitudinal section tangential to the vascularcylinder. A large portion of the sieve tube on the left goes out of the section plane (at the arrowhead). Numbering of thesieve elements starts with the so-called sieve plate 1-1' and proceeds upwards and downwards (cf. also Fig. 2). A scale infim has been adjusted on the left of the figure to indicate the distance of each cell from the root apex (zone of contactbetween the apical meristem and the calyptrogen). X310.Fig. 2. The sieve plate 1-1', situated between the last differentiating sieve element bearing ribosomes (SE 1) and the firstone deficient in stainable contents (SE 1'), that provides a reliable starting point for protophloem sieve element numbering.X5300.

Microtubules and sieve element wall in wheat 597

ascribed to them in the sieve element-companion cell wall thickening is brought about by contributions ofcomplexes (Gunning, 1976; Esau & Thorsch, 1985). wall material from the sieve element only.On the companion cell side the cell wall remains During the stages of intensive wall deposition (SEssmooth and unthickened. The asymmetrical position of 6—2) MTs along the lateral walls of sieve elementsthe middle lamella (Figs 9, 10, 12) clearly indicates that (Figs 7-10, 12) are abundant, there being more than

mma

mmm-598 £. P. Eleftheriou

10 MTs [im '. While the majority of MTs are peripher-ally distributed and oriented perpendicularly to thelongitudinal axis, a minority are encountered in innerpositions, close to the nucleus, the mitochondria or freein the cytoplasm (Fig. 13). Aberrant MTs are mostfrequently found in the older cells of the vertical series(SEs 3-1), and do not show a definite orientation.

What is peculiar about differentiating sieve elementsof wheat is that MTs are not spaced out evenly alongthe whole wall surface: they specifically overlie devel-oping regions, while they are scarce or, most usually,entirely absent from the narrow non-thickened areasoccupied by plasmodesmata (Figs 8-10, 12-14).Graphical expression of the scarcity and abundance ofMTs along the thickened and non-thickened areas in anSE 2 is characterized by deep narrow depressionsalternating with broad ridges; the depressions corre-spond to non-thickened areas, the ridges to thickenedones. Measurement of the extent occupied by the twowall areas in SEs 2 revealed thickened regions occupy-ing 82-8% of the whole wall length and the non-thickened ones 11 -2%. The average densities of MTswere found to be 12-2MTs/xm~l in the thickenedregions and 0-5 MTs //m~' in the non-thickened ones,corresponding to a percentage of 99-2% and 0-8%,respectively.

Sections cutting the lateral walls obliquely revealMTs running parallel to each other and confirm theabsence of MTs from the cytoplasm within the de-pressions (Fig. 14). A major feature visible in suchplanes of section is the correspondence between theorientation of MTs and that of cellulose microfibrils inthe wall (Figs 14, 15).

Longitudinal sections tangential to the cell surfaceshow MTs oriented approximately parallel to eachother and perpendicular to the long axis of the cell(Figs 15, 16). A few MTs, however, deviate by largeror smaller angles relative to the main arrays (Figs 15,16, arrows), while some are vertically oriented

Fig. 3. Nascent cell wall between an SE 15 and itscompanion cell (cc). A few MTs are spaced out along thewall on the sieve element side. MTs are also found in thevicinity of plasmodesmata (arrows). The encircled MTs arebridged to the plasma membrane. X46900.Figs 4, 5. SE 12 (Fig. 4), SE 9 (Fig. 5). There is a slightincrease in the occurrence of MTs along the wall on thesieve element side, but now they can hardly be observedclose to plasmodesmata. The latter are encountered insingle or in couples (arrows). Both: X46900.Fig. 6. SE 7. MTs are evenly distributed along the wholelateral wall, which is yet thin. Note the 'dilute' appearanceof the cytoplasm in the sieve element compared to that ofthe companion cell (cc). x46900.Fig. 7. SE 6. Initial stage of the non-uniform cell wallthickening. The developing thickenings are underlain bynumerous MTs, which are absent from the non-thickeningsites (areas marked by brackets), x46900.

(Fig. 16, arrowhead). A closer examination of serialsections (such as those of Figs 15, 16) indicates thatthis microheterogeneity occurs in a region of thecortical cytoplasm somewhat deeper than that of themain arrays of MTs. The appearance of MTs in Fig. 13provides additional support for this suggestion.

Numerous vesicles are found amongst the MTs.They form two populations: small, densely stainingvesicles, and bigger ones with fibrillar/granular con-tent. Some coated vesicles are also observed (Figs 15,16). The least spacing between adjacent MTs wascalculated to be 22-4±6-5nm; the outer diameter ofthe small vesicles was found 64-7 ±5-7 nm, and that ofthe big ones 128-8 ± 20-7 nm (in all: n = 40, where n isthe sample size; ± = standard deviation).

Discussion

The peculiar wall thickenings

The developmental features of protophloem sieve el-ements in roots have been studied ultrastructurally in anumber of plant species: Phaseolus vulgaris (Esau &Gill, 1971), Nicotiana tabacum (Esau & Gill, 1972),Allium cepa (Esau & Gill, 1973), Lemna minor (Mel-aragno & Walsh, 1976), Hordeum vulgare (Danilova &Telepova, 1978), Isoetes muricata (Kruatrachue &Evert, 1978), Oryza saliva (Kawahara et al. 1980),Gossypium hirsutum (Thorsch & Esau, 1981), Aegilopscomosa (Eleftheriou & Tsekos, 1982) and Salixviminalis (Fjell, 1986). Uneven wall thickenings simi-lar to those observed in this study in wheat have beendescribed in the counterpart cells of Hordeum vulgare(Danilova & Telepova, 1978) and Aegilops comosa(Eleftheriou & Tsekos, 1982). They can also be clearlyseen in rice (Kawahara et al. 1980), but hardly at all inother monocotyledons and all other species of the abovelist. Though the number of plant species investigated issmall, it should be pointed out that these peculiar wallthickenings have been encountered to date only inspecies belonging to the family Poaceae. Should that beproved correct it would constitute a unique ultrastruc-tural characteristic of the members of Poaceae, whichmight be significant for plant cytologists and taxo-nomists.

In the studies cited in the preceding paragraph quitedifferent ways of sieve-element labelling have beenused, in each case depending on a good longitudinalsection. The numbering system described here seemsto have an advantage over the variety of methods usedto date, in that it depends on a readily recognizable andreliable starting point for numbering, it is very simpleto use, it might compromise the existing variantnumbering systems, it is unrelated to the sieve tubelength in ultrathin sections, and, since an abrupt

Microtubules and sieve element wall in wheat 599

k&

Figs 8—10. SEs 5, 4, 3. Successive stages of the non-uniform wall thickening that progresses on the sieve element sideonly. The asymmetrical position of the middle lamella (ml) clearly marks the wall components contributed by the twopartners. MTs are again missing from the non-thickened areas (brackets). ER elements (arrows) are associated with theplasmodesmata. All: X46900.Fig. 11. A low magnification view of the side wall of an SE 2 showing its highly irregular thickenings. X6400.

600 E. P. Eleftheriou

***&%*i N ' *

Fig. 12. Details of an SE 2. Uneven wall development creates narrow depressions and broad ridges. MTs underlie theridges only. Many vesicles are encountered adjacent to the plasma membrane (arrows), in, mitochondria; p, plastids;ml, middle lamellae. X46 100.Fig. 13. A two-piece figure of an SE 1: between the upper and lower parts (at the rule) there is a missing portion of about1/im. Besides the cortical MTs, additional ones can be seen free in the cytoplasm (filled arrows), close to mitochondria (m)(arrowhead) or to the nucleus (n) (open arrow). X47 100.

Microtubules and sieve element wall in wheat 601

change is also observed in the sieve tubes of the otherspecies investigated, it is universally applicable.

The secondary wall thickenings of differentiatingxylem elements occur as distinct bands (Pickett-Heaps,1968; Hepler, 1981). In the two-dimensional micro-graphs the developing walls of sieve elements of wheatappear to consist of growing ridges alternating withdepressions. In both xylem and sieve elements group-ings of MTs predict areas of wall thickening, anddeveloping thickenings indeed begin and proceed be-neath the groups of MTs; the intervening gaps aremarked by the absence of MTs. These similarities raisethe possibility that similar mechanisms are at work insieve elements and xylem cell development.

A number of differences between the two cell typesshould not, however, be overlooked. In sieve elementsthe non-thickened areas are occupied by plasmodes-mata, while it is not clear whether the ridges are indeedbands in the sense of xylem elements; although con-vincing evidence is missing at present, they are mostprobably patches surrounding islands of grouped plas-modesmata. ER cisternae are encountered in both celltypes. In sieve elements ER is associated with plasmo-desmata, a normal condition, not only for sieve el-ements (Gunning, 1976; Esau & Thorsch, 1985) butalso for all cell types (Robards, 1976); in xylem

elements ER cisternae, being interspersed between thebands of MTs and lying flat against the wall, wereassumed to be involved in the pattern of wall develop-ment by preventing access of vesicles to the underlyingplasma membrane, thus ensuring that wall material isdelivered to the actual thickenings (Pickett-Heaps,1967, 1968; Pickett-Heaps & Northcote, 1966).

But what might the function of these peculiarthickenings be? While in the xylem elements secondarythickenings are permanently deposited, there is goodevidence that in sieve elements the thickenings arereversible, since enucleate cells gradually obtainsmooth walls (Eleftheriou & Tsekos, 1982). Danilova &Telepova (1978) presented evidence suggesting that thethickenings serve as stores of excess wall material that issynthesized profusely during the active stages of celldifferentiation. The excess of wall material and theplasma membrane lining it are used in subsequentgrowth of the enucleate stage when most cytoplasmiccomponents of the sieve elements - including the Golgiapparatus - have undergone autolytic destruction,rendering the production of new wall and membranematerial impossible. At the enucleate stage sieve el-ements are known to elongate, being passivelystretched by the surrounding cells, which elongateactively. If the enucleate sieve elements are to keep

.-V- -•.-.!.*•«.-

^ - ,

Fig. 14. Obliquely sectioned lateral wall of an SE 1. MTs overlie the wall thickenings only; their orientation corresponds tothat of cellulose microfibrils (double-headed arrow), cc, companion cells; p, plastids; m, mitochondria. X36 100.

602 E. P. Eleftheriou

M*i

ISp^lSmsiFigs 15, 16. Two serial sections of exactly the same area of an SE 3, tangential to the cell surface. The section of Fig. IS isthe outermost one, that of Fig. 16 is deeper towards the cytoplasm. The majority of MTs are transversely aligned, some areoriented at slight or greater angles (arrows) relative to the main groups, while a few run vertically (arrowheads). Numerousvesicles classified in two populations abound amongst the MTs. The orientation of MTs is mirrored by that of cellulosemicrofibrils (Fig. 15, double-headed arrow), cv, coated vesicles. Both: X40000.

Microtubules and sieve element wall in wheat 603

pace with this rapid elongation and avoid crushing andobliteration they have to increase their surface area.Extension of the surface can be effected by redistri-bution of stored material.

The densities of tnicrotubules

Some analogies exist between sieve elements of wheatand xylem elements, concerning features of the MTs.Both in the very young sieve elements of wheat (SEs16-12) and in the xylem initials olAzolla (Hardham &Gunning, 1979) the cortical MTs are distributedevenly along the longitudinal walls, as they are in theincipient xylem cells of wheat coleoptiles (Pickett-Heaps, 1974). At a stage before any formation ofsecondary thickenings in xylem elements the MTsbecome grouped into bands. Grouping is accompaniedby an increase in the number of MTs nm~'. The steadyincrease in MT densities in sieve elements of wheat(SEs 12-7) occurs at about the same developmentalstage as in xylem elements of Azolla, namely before theinitiation of wall thickening.

15—1

14-

6 -

5 -

4 -

3 -

2 -

1-

In differentiating sieve elements MTs are encoun-tered at great densities. As is the case with the sieveelements of Azolla (Hardham & Gunning, 1979) andcotton roots (Thorsch & Esau, 1982), the maximumnumbers of MTs in wheat (up to 14 ^m~') wereobserved when the increase in wall thickening was mostobvious (SEs 7-2), their average number being greaterthan 10/zm"1 (Fig. 17). In Azolla roots the rate ofproduction of MTs per cell was estimated to be muchhigher in sieve elements than in other cell typesinvestigated - including xylem elements - and theiraverage densities adjacent to the walls of differentiatingsieve elements (12 MTsjUm"1) were the highest amongall cell types studied, being 50% higher than in thexylem elements themselves (S-OMTs^im"1) (Hard-ham, 1982).

Being the first cell type to differentiate in the apicallygrowing roots, protophloem sieve elements reachmaturity very close to the root apex (see Esau, 1969,p. 180). In fact the span between initiation of wallmaterial deposition and completion (SEs 6-1), being

17 16 15 14 13—r12 n

"T"10

T"

rSieve element position in the protophloem file

Fig. 17. Distribution of MTs along the lateral walls of developing protophloem sieve elements of wheat. Each symbolrepresents an average calculated from one longitudinal wall. The graph can be divided into three phases: a steady increase(SEs 16—8), a plateau phase (SEs 7-2), and a sharp decline (SE 1).

604 E. P. Eleftheriou

about 220 ̂ im long, coincides completely with themaximum numbers of MTs. In addition, the sharpdecline in MT numbers in SE 1 coincides with theculmination of the degrading differential processes ofthe cell, including the most dynamic stages of nucleardegeneration (Eleftheriou, 1987). These correlationsstrongly support the view that MT densities reflect therate of wall material production and deposition of sieveelements. Although MTs are neither involved in thesynthesis of cellulose nor do they influence the quantityof synthesis (Grimm et al. 1976), they apparently playa direct and active role in sieve element morphogenesis,presumably by imposing spatial control on cell wallformation, thereby helping to determine the shape thatis assumed by the expanding cell (cf. Marchant, 1979;Gunning & Hardham, 1982).

The details of the rise and fall of MT densities indifferentiating sieve elements of wheat are, however,not in full agreement with those of Azolla (Hardham &Gunning, 1979) and cotton (Thorsch & Esau, 1982)sieve elements. The first investigators estimated andexpressed graphically the number of MT profilesper^rn along the longitudinal walls of differentiatingsieve elements, distinguishing between young andolder roots as they bear longer and shorter merophytes,respectively. The graph depicting the densities of MTsin wheat (Fig. 17) parallels that of the young roots ofAzolla as regards the increase and maximum phasesonly; the decline phase in wheat falls much moreabruptly. In cotton roots (Thorsch & Esau, 1982) themaximum number of MTs were present during 'latestage 1'. The authors defined this condition as the'plateau phase'. The plateau phase of cotton corre-sponds to SEs 8 and 7 of wheat. A plateau phase canalso be defined in wheat, but it occurs in SEs 7-2,corresponding to about stage 2 of cotton differentiatingsieve elements.

Microtubules and microfibril orientationThe MT-microfibril parallelism in sieve elements ofwheat is in good agreement with other observations onexpanding cells (Marchant, 1979; Gunning & Hard-ham, 1982), as well as with the conclusions of recentstudies on MTs using immunofluorescence techniques,which confirmed the existence of a relatiorl between cellexpansion and MT orientation (Wicked al. 1981; Traaset al. 1984; Derksen et al. 1986). Cell expansion andco-alignment of MTs and cellulose microfibril depo-sition are related phenomena (Derksen et al. 1986).

In sieve elements of wheat, however, some MTsdeviate from the perpendicular alignment. AberrantMTs have also been seen in other cell types (Hepler &Fosket, 1971; Pickett-Heaps, 1974; Hardham & Gun-ning, 1978, 1979; for review, see Robinson & Quader,1982). The aberrant MTs do not conform with thegenerally accepted view of congruence in orientation of

cortical MTs and currently deposited microfibrils inthe wall (Newcomb, 1969; Hepler & Palevitz, 1974;Hepler, 1981; Gunning & Hardham, 1982). Pickett-Heaps (1974), observing bands of MTs between slightregular ridges of differentiating xylem walls, believesthey are a temporary condition. The significance of theaberrant MTs, if any, is not clear and needs furtherexperimental and structural documentation. Thus, thequestion of how the MTs are properly aligned is notanswered. Even if we explain how MTs control theoriented deposition of cellulose the question of how theMTs themselves become aligned is left open (Gunning,1981).

Microtubules and Golgi vesicles

During deposition of the localized thickenings in xylemelements MTs overlying them have been supposed todirect the Golgi apparatus-derived vesicles to thegrowing thickenings (Pickett-Heaps, 1967, 1968; New-comb, 1969; Maitra & De, 1971). It has converselybeen pointed out that MTs are so close to one anotherthat intervening spaces are insufficient to allow objectsof the size of Golgi vesicles to pass between them and soto reach the membrane surface (Goosen-de Roo,1973a,b). This hypothesis does not seem justified insieve elements of wheat, where the associations be-tween MTs and Golgi vesicles are so frequent andintimate. Though the spaces between adjacent MTs(22-4 ± 6-5 nm) are actually much smaller than thediameter of even the small-sized vesicles (64-7 ±5-7 nm), both kinds of vesicles were encountered at thesame level as MTs, and interspersed among them(Figs 15, 16). In other species, Golgi vesicles amongstthe MTs of sieve elements have been demonstrated inleaves of Aegilops (Eleftheriou & Tsekos, 1982), andwere observed close to MTs of Azolla (Hardham &Gunning, 1979), sugarcane (Singh, 1980) and cotton(Thorsch & Esau, 1982) differentiating sieve elements.It seems that MTs, being highly labile structures(Newcomb, 1969), not only do not act as a barrier buteven effect the movement of vesicles through theirarrays, presumably by subjecting them to reversibleslight sidewards movements. Galatis (1980) suggestedthat MTs may be involved in Golgi vesicle distributionalong the protoplast surface, presumably in that wayaffecting the pattern of the wall thickening.

The author is thankful to Mrs A. Kiratzidou-Dimopouloufor skilful secretarial assistance, and to the Stiftung Volks-wagenwerk for financial support.

References

BEHNKE, H.-D. & DORR, I. (1967). Zur Herkunft undStruktur der Plasma-filamente in Assimilatleitbahnen.Planta 74, 18-44.

Microtubules and sieve element wall in wheat 605

BOUCK, G. B. & CRONSHAW, J. (1965). The fine structureof differentiating sieve tube elements. J . Cell Biol. 25,79-96.

CRONSHAW, J. & BOUCK, B. G. (1965). The fine structureof differentiating xylem elements. J. Cell Biol. 24,415-431.

DANILOVA, M. F. & TELEPOVA, M. N. (1978).

Differentiation of protophloem sieve elements in seedlingroots of Hordeum vulgare. Phytomorphology 28, 418-431.

DERKSEN, J., JEUCKEN, G., TRAAS, J. A. & VAN

LAMMEREN, A. A. M. (1986). The microtubular skeletonin differentiating root tips of Raphanus sativus L. Actabot.neerl. 35, 223-231.

DOOHAN, M. E. & PALEVTTZ, B. A. (1980). Microtubules

and coated vesicles in guard cell protoplasts of Alliutncepa L. Plartta 149, 389-401.

DUTE, R. R. & EVERT, R. F. (1977). Sieve-element

ontogeny in the root of Equisetum hyemale. Am. jf. Bot.64, 421-438.

ELEFTHERIOU, E. P. (1985). Microtubules and rootprotophloem ontogeny in wheat. J. Cell Sci. 75,165-179.

ELEFTHERIOU, E. P. (1987). Ultrastructural studies onprotophloem sieve elements in Triticum aestivwn L.Nuclear degeneration.^. Ultrastruct. molec. Struct. Res.(in press).

ELEFTHERIOU, E. P. & TSEKOS, I. (1982). Developmental

features of cell wall formation in sieve elements of thegrass Aegilops contosa var. thessalica. Ann. Bot. 50,519-529.

ESAU, K. (1969). The Phloem. In Encyclopedia of PlantAnatomy, vol. 2, (ed. W. Zimmermann, P. Ozenda & H.D. Wulff) p. 180. Berlin, Stuttgart: GebruderBorntraeger.

ESAU, K. (1978). Developmental features of the primaryphloem in Phaseolus vulgaris L. Ann. Bot. 42, 1-13.

ESAU, K. & GILL, R. H. (1971). Aggregation ofendoplasmic reticulum and its relation to the nucleus in adifferentiating sieve element, jf. Ultrastruct. Res. 34,144-158.

ESAU, K. & GILL, R. H. (1972). Nucleus and endoplasmicreticulum in differentiating root protophloem ofNicotiana tabacum.J. Ultrastruct. Res. 41, 160-170.

ESAU, K. & GILL, R. H. (1973). Correlations indifferentiation of protophloem sieve elements of AJliumcepa root. J. Ultrastruct. Res. 44, 310-328.

ESAU, K. & THORSCH, J. (1985). Sieve plate pores andplasmodesmata, the communication channels of thesymplast: ultrastructural aspects and developmentalrelations. Am. J. Bot. 72, 1641-1653.

EVERT, R. F. (1976). Some aspects of sieve-elementstructure and development in Botrychium virginianum.Israel J. Bot. 25, 101-126.

FJELL, I. (1986). Ultrastructural features of differentiatingprotophloem sieve elements in adventitious roots of Saltxviminalis. Nord. J. Bot. (in press).

GALATIS, B. (1980). Microtubules and guard-cellmorphogenesis in Zea mays L. J. Cell Sci. 45, 211-244.

GIDDINGS, T. H., BROWER, D. L. & STAEHELIN, L. A.

(1980). Visualization of particle complexes in the plasmamembrane of Micrasterias denticulata associated with

the formation of cellulose fibrils in primary andsecondary cell walls. J. Cell Biol. 84, 327-339.

GREEN, P. B. (1980). Organogenesis - a biophysical view.A. Rev. PL Physiol. 31, 51-82.

GRIMM, I., SACHS, H. & ROBINSON, D. G. (1976).

Structure, synthesis and orientation of microfibrils.II. The effects of colchicine on the wall of Oocystissolitaria. Cytobiologie 14, 61-74.

GOOSEN-DE ROO, L. (1973a). The relationship betweencell organelles and cell wall thickenings in primarytracheary elements of the cucumber. I. Morphologicalaspects. Acta bot. need. 22, 279-300.

GOOSEN-DE ROO, L. (19736). The relationship betweencell organelles and cell wall thickenings in primarytracheary elements of the cucumber. II. Quantitativeaspects. Acta bot. neerl. 22, 301-320.

GUNNING, B. E. S. (1976). The role of plasmodesmata inshort distance transport to and from the phloem. InIntercellular Communication in Plants: Studies onPlasmodesmata (ed. B. E. S. Gunning & A. W.Robards), pp. 203-227. Berlin, Heidelberg, New York:Springer-Verlag.

GUNNING, B. E. S. (1981). Microtubules andcytomorphogenesis in a developing organ: The rootprimordium of Azolla pinnata. In Cytomorphogenesis inPlants (ed. O. Kiermayer), Cell Biol. Monographs, vol.8, pp. 301-325. Wien, New York: Springer-Verlag.

GUNNING, B. E. S. & HARDHAM, A. R. (1982).

Microtubules. A. Rev. PL Physiol. 33, 651-698.HARDHAM, A. R. (1982). Regulation of polarity in tissues

and organs. In The Cytoskeleton in Plant Growth andDevelopment (ed. C. W. Lloyd), pp. 377-403. London,New York: Academic Press.

HARDHAM, A. R. & GUNNING, B. E. S. (1978). Structure

of cortical microtubule arrays in plant cells. J. Cell Biol.77, 14-34.

HARDHAM, A. R. & GUNNING, B. E. S. (1979).

Interpolation of microtubules into cortical arrays duringcell elongation and differentiation in roots of Azollapinnata. J. Cell Sci. 37, 411-442.

HEPLER, P. K. (1981). Morphogenesis of trachearyelements and guard cells. In Cytomorphogenesis in Plants(ed. O. Kiermayer), Cell Biology Monographs, vol. 8,pp. 327-347. Wien, New York: Spnnger-Verlag.

HEPLER, P. K. & FOSKET, D. E. (1971). The role of

microtubules in vessel member differentiation in Coleus.Pmtoplasma 72, 213-236.

HEPLER, P. K. & PALEVTTZ, B. A. (1974). Microtubules

and microfibrils. A. Rev. PI. Physiol. 25, 309-362.KAWAHARA, H., MATSUDA, T. & CHONAN, N. (1980).

Studies on morphogenesis in rice plant. XII.Ultrastructure of the phloem in the crown root. (Jap.).Jap.J. Crop Sci. 49, 330-339.

KRUATRACHUE, M. & EVERT, R. F. (1978). Structure and

development of sieve elements in the root of Isoetesmuricata Dur. Ann. Bot. 42, 15-61.

LEDBETTER, M. C. & PORTER, K. R. (1963). A

"microtubule" in plant cell fine structure. J. Cell Biol.19, 239-250.

606 E. P. Eleftheriou

MAURA, S. C. & D E , D. N. (1971). Role of microtubulesin secondary thickening of differentiating xylem element.J. Ultrastruct. Res. 34, 15-22.

MARCHANT, H. J. (1979). Microtubules, cell walldeposition and the determination of plant cell shape.Nature, bond. 278, 167-168.

MELARAGNO, J. E. & WALSH, M. A. (1976).

Ultrastructural features of developing sieve elements inLemna minor L. - The protoplast. Am. J. Bot. 63,1145-1157.

NEWCOMB, E. H. (1969). Plant microtubules. A. Rev. PI.Physiol. 20, 253-288.

PALEVTTZ, B. A. (1982). The stomatal complex as a modelof cytoskeletal participation in cell differentiation. In TheCytoskeleton in Plant Growth and Development (ed. C.W. Lloyd), pp. 345-376. London, New York: AcademicPress.

PICKETT-HEAPS, J. D. (1967). The effects of colchicine onthe ultrastructure of dividing plant cells, xylem walldifferentiation and distribution of cytoplasmicmicrotubules. Devi Biol. 15,206-236.

PICKETT-HEAPS, J. D. (1968). Xylem wall deposition.Radioautographic investigations using lignin precursors.Protoplasma 65, 181-205.

PICKETT-HEAPS, J. D. (1974). Plant microtubules. InDynamic Aspects of Plant Ultrastructure (ed. A. W.Robards), pp. 219-255. Maidenhead, Berkshire,England: A. W. McGraw-Hill.

PICKETT-HEAPS, J. D. & NORTHCOTE, D. H. (1966).

Relationship of cellular organelles to the formation anddevelopment of plant cell wall. jf. exp. Bot. 17, 20—26.

ROBARDS, A. W. (1976). Plasmodesmata in higher plants.In Intercellular Communication in Plants: Studies onPlasmodesmata (ed. B. E. S. Gunning & A. W.Robards), pp. 15-53. Berlin, Heidelberg, New York:Springer-Verlag.

ROBINSON, D. G. & QUADER, H. (1982). The microtubule-

microfibril syndrome. In The Cytoskeleton in PlantGrowth and Development (ed. C. W. Lloyd), pp.109-126. London: Springer-Verlag.

SINGH, A. P. (1980). On the ultrastructure anddifferentiation of the phloem in sugarcane leaves.Cytologia 45, 1-31.

SJOLUND, R. D. & SHIH, C. Y. (1983). Freeze-fracture

analysis of phloem structure in plant tissue cultures.II. The sieve element plasma membrane. J. Ultrastruct.Res. 82, 189-197.

TAMULEVICH, S. R. & EVERT, R. F. (1966). Aspect of sieveelement ultrastructure in Primula obconica. Planta 69,319-337.

THORSCH, J. & ESAU, K. (1981). Ultrastructural studies ofprotophloem sieve elements in Gossypium hirsutum. J.Ultrastruct. Res. 75, 339-351.

THORSCH, J. & ESAU, K. (1982). Microtubules in

differentiating sieve elements of Gossypium hirsutum. jf.Ultrastruct. Res. 78, 73-83.

TRAAS, J. A., BRAAT, P. & DERKSEN, J. (1984). Changes

in microtubule arrays during the differentiation ofcortical root cells of Raphanus sativus. Eur. J. Cell Biol.34, 229-238.

WICK, S. M., SEAGULL, R. W., OSBORN, M., WEBER, K. &

GUNNING, B. E. S. (1981). Immunofluorescencemicroscopy of organized microtubule arrays instructurally-stabilized meristematic plant cells, jf. CellBiol. 89, 685-690.

WOODING, F. B. P. & NORTHCOTE, D. H. (1964). The

development of the secondary wall of the xylem of Acerpseudoplatanus. J. Cell Biol. 23, 327-337.

(Received 24 November 1986 -Accepted, in revised form,3 March 1987)

Microtubules and sieve element wall in wheat 607