The Plant Cell, Vol. 6, 1241-1252, September 1994 O 1994 American Society of Plant Physiologists

Endosperm Development in Barley: Microtubule lnvolvement in the Morphogenetic Pathway

Roy C. Brown,a Betty E. Lemmon,a and OddArne Olsen bi‘

a Department of Biology, University of Southwestern Louisiana, Lafayette, Louisiana 70504-2451 Plant Molecular Biology Laboratory, Agricultura1 University of Norway, PO. Box 5051, N-1432 As, Norway

An immunofluorescence study of sectioned barley endosperm imaged by confocal laser scanning microscopy provided three-dimensional data on the relationship of microtubules to the cytoplasm, nuclei, and cell walls during development from 4 to 21 days after pollination (DAP). Microtubules play an important role throughout endosperm ontogeny. The syn- cytium is organized into units of nuclear-cytoplasmic domains by nuclear-based radial microtubule systems that appear to control the pattern of the first anticlinal walls at 5 to 6 DAP. After 7 DAP, phragmoplasts of two origins (interzonal and cytoplasmic) guide wall formation. Large compartments formed by the “free growing” walls in association with cyto- plasmic phragmoplasts formed adventitiously at interfaces of opposing microtubule systems are subsequently subdivided by interzonal phragmoplastlcell plates to give rise to the starchy endosperm. During development of the aleurone layer from 8 to 21 DAP, the microtubule cycle is typical of plant histogenesis; cortical microtubules are hooplike, and preprophase bands of microtubules predict the division plane.

INTRODUCTION

In spite of the enormous economic importance of cereal en- dosperm in agriculture and industry, surprisingly little is known about its development and evolution. The endosperm is an enigma in that (1) it is a product of double fertilization &e., the union of one sperm cell with the egg results in the diploid zy- gote and the other sperm with one or more nuclei results in the primary endosperm nucleus); (2) unlike the embryo, which follows generally recognized patterns of plant histogenesis, the endosperm is so unlike any other plant tissue that it has been called a monstrosity; and (3) the endosperm has proven extremely difficult to study because in its early stage, it is a delicate liquid coenocyte contained within the innermost cham- ber of the developing seed.

The evolutionary introduction of the endosperm into the life cycle of angiosperms is unknown. Double fertilization occurs in gymnosperms (Friedman, 1992), but the second fertiliza- tion results in an additional embryo that competes for resources during development. In angiosperms, the second fertilization results in the primary endosperm nucleus from which develops the nutritive tissue. The endosperm could either be a delayed continuation of female gametophyte development triggered by the second fertilization event and therefore homologous to the nutritive gametophyte in gymnosperms (Thomas, 1907), or the endosperm may be a highly modified supernumerary embryo (Sargant, 1900). Support for the second hypothesis is amassed by Friedman (1994), who argues that the endosperm is a homo- log of the embryo, or in other words, an organism in its own

To whom correspondence should be addressed.

right. Recent molecular data demonstrating the expression of the same or homologous genes in the embryo and endosperm (Aalen et al., 1994) lend support to this view. From this per- spective, the endosperm emerges as a model system for the study of plant development. It is abundant, fast growing, and consists of only two cell types that can be mechanically sepa- rated (Jakobsen et al., 1989), and importantly, genetic mutants affecting the development of the endosperm are known (Sheridan and Neuffer, 1980; Felker et al., 1985; Bosnes et al., 1987, 1992; Kowles et al., 1992).

Three general types of endosperm development are recog- nized (Brink and Cooper, 1947; Vijayraghavan and Prabhakart, 1984; Friedman, 1994): ab initio cellular, in which all mitoses are followed by wall development; nuclear, in which free nu- clear divisions result in a syncytium that later becomes cellularized; and helobial, where the first division divides the central cell into separate micropylar and chalazal chambers that develop independently. Cereal grains undergo the nuclear type of development with cell walls forming in the immediate vicinity of the developing embryo region (micropylar) earlier than those in the large chalazal region (Engell, 1989). In all but the restricted area near the embryo, a coenocytic stage is followed by cellularization, differentiation into starchy en- dosperm and aleurone, and maturation by engorgement with storage compounds and dehydration (Olsen et al., 1992).

Endosperm development in cereal grains seems to have much in common with nuclear endosperm development in other systems, suggesting that the process of endosperm de- velopment is relatively conserved. The cellular and molecular

1242 The Plant Cell

mechanisms involved in the organization and cellularization in early endosperm are complex and relatively little understood. At least four distinct types of wall development have been rec- ognized (Newcomb, 1973: Newcomb and Fowke, 1973; Olson, 1981; Ponzi and Piuolongo, 1984; Gori, 1987; Yeung and Cavey, 1988): (1) labyrinthine walls of the transfer type (Pate and Gunning, 1972) that protrude from the embryo sac wall into the central cell early in development are thought to be partic- ularly important in supplying nutrients to the young embryo through loading of metabolites from the surrounding nucellus; (2) free-growing walls grow as fingerlike projections from the embryo sac wall into the central vacuole. These walls, which are the first indication of cellularization, are independent of mitotic spindles and do not appear to be initially associated with microtubules or microfilaments; (3) cell plates associated with typical phragmoplasts arise in interzonal regions of mi- toticfigures, as istypical of plant cytokinesis; and (4) in addition to the above-mentioned wall types that partition the cytoplasm, thin walls may develop at the surface of the coenocytic cytoplasm adjacent to the central vacuole.

All of the above have been reported at one time or another for cereal grains. Although earlier studies reported that initial walls grow freely in the absence of phragmoplasts (Morrison and OBrien, 1976) or with phragmoplast-like configurations in the tips of wall that develop in fingerlike extensions of the cytoplasm projecting into the central vacuole (Mares et al.,

19751, Fineran et al. (1982) concluded that in wheat ali walls actually grow in association with phragmoplasts, as is typical of plant cytokinesis. However, as pointed out by van Lammeren (1988), the question of how phragmoplasts develop between non-sister nuclei remains unanswered.

The goal of this study isto provide a developmental history of barley endosperm based on the organization of microtubules as evidence of growth and morphogenesis. A combination of techniques was used to study in situ development of the en- dosperm in carefully staged barley grains that were grown under uniform conditions in a phytotron. In this article, we pre- sent three-dimensional information on the relationships of microtubules to organization of the cytoplasm and placement of walls that demonstrates the prominent role of the micro- tubular cytoskeleton in development of the endosperm.

RES U LTS

In this study, we describe the organization of microtubules and correlated aspects of wall development in endosperm of bar- ley from 4 to 21 days after pollination (DAP). These data are summarized (Figure 1) in relationship to the time course for developmental events established by Bosnes et ai. (1992).

I Syncytial

I1

I11 Differentiation

DAP 4 8 12 16 20 I I I I I I I I I I I I I I I I I I I I

I Syncytial mitosis without cytokinesis 0 Vacuolation

0 Cytoplasmic shaping A

I1

I11

Free wall formation 8 Cytoplasmic phragmoplasts 4 Interzonal phragmoplasts B m

Subdivision of starchy endosperm

Aleurone mitosis 0 Cortical arrays and PPB in aleurone meristem C

Figure 1. Stages and Timing of Main Events of Barley Endosperm Morphogenesis.

The time course expressed in days after hand pollination (DAP) for the first three stages of endosperm development (maturation is excluded), as described by Bosnes et al. (1992), is shown in the top three bars. The lower bars show the main morphogenetic events based on the immunoflu- orescence study of microtubules and cell walls reported in this study. Symbols represent interphase microtubule systems characteristic of each of the major stages. (A) Nuclear-based radial microtubules in the syncytial stage. (6) Modified system of nuclear-based microtubules during formation of initial anticlinal walls. (C) Hooplike cortical microtubules and PPB during aleurone development.

Microtubules in Endosperm of Barley 1243

The barley ovule is teardrop shaped with the developing em-bryo positioned in the pointed (proximal) tip (Figure 2A). Thesomewhat flattened grain is bilaterally symmetrical with a cen-tral groove on the ventral side (Figure 2B). The micrographspresented in Figures 3 to 7 resulted from transverse and lon-gitudinal sections through the midportion of the ovule. Allmicrographs, except tangential sections, are oriented with thecentral vacuole toward the top.

Syncytial Stage

The early endosperm is a multinucleate syncytium that de-velops from the primary endosperm nucleus via a seriesof mitotic divisions without cytokinesis. At 4 DAP, waves ofnearly synchronous mitoses (Figures 3A and 3B) continueto increase the number of nuclei in the syncytium. The mitoticapparatus is typical of plant cell division with broad spindlepoles comprising numerous foci of microtubules (minipoles).The metaphase spindle (Figure 3B) is comprised of bundlesof kinetochore microtubules and nonkinetochore microtubulesthat appear to branch from them. The nonkinetochore micro-tubules interconnect the kinetochore fiber complexes withinand between the half spindles. In anaphase (Figure 3B),kinetochore bundles shorten and nonkinetochore bundlesoverlap in the interzone. In telophase (Figure 3B), microtubulesproliferate around the two groups of daughter chromosomes;those emanating from the poles form asters (not present inearlier stages), and those on opposing surfaces interact in theequatorial region to give rise to a distinct interzonal array. Theinterzonal array increases in structural organization until a dis-tinct columnlike phragmoplast further separates the nuclei(Figures 3A and 3C). However, the phragmoplasts are shortlived and do not function in deposition of a cell plate leadingto cytokinesis. Instead, the phragmoplasts break down, leavingbrightly fluorescent fringes on the proximal sides of the daugh-ter nuclei, as shown in Figure 3A, and radial microtubulesemanating from the nuclei interconnect neighboring nuclei(Figure 3C). The nuclei become evenly spaced in the cytoplasmin association with the development of radial microtubule sys-tems (RMSs) that emanate equally from interphase nuclei inthe syncytium (Figure 3D). The RMSs appear to maintainspacing of nuclei in the peripheral syncytium and serve todefine domains of cytoplasm around each nucleus (Figure 3D).Microtubules of opposing radial systems overlap one another(Figure 3E), and the cytoplasm remains undivided until ~5to 6 DAP. At this time, numerous microtubules clearly definethe nuclear cytoplasmic domains (NCDs), and wall materialis deposited in the zones of interaction between opposingarrays (Figure 3F).

Cellularization

Vacuolation, shaping of the cytoplasm, and deposition of thefirst anticlinal walls mark the transition from syncytial to cellular

Figure 2. Scanning Electron Micrographs of 3-DAP Barley Ovules.

(A) Isolated ovule with the micropylar tip lowermost. Magnification x30.(B) Ovule sectioned to show the arrangement of the central vacuole(CV) surrounded by the thin layer of syncytial endosperm (SE) withinthe nucellus and vascular tissue (V) in the ventral crease. Magnifica-tion x45.

endosperm (Figure 1). The frequency of mitotic division dimin-ishes during the period of initial wall development, and nophragmoplasts were observed that would be in position to ini-tiate the anticlinal walls at 5 to 6 DAP. Therefore, it appearsthat a unique type of wall development occurs during initialcompartmentalization of the central cell. These walls, termedfree-growing walls in earlier literature, form at the boundariesof NCDs (Figure 4A, compare to microtubules in Figure 3F)and grow into the central vacuole from the peripheral syncyliumto subdivide the cytoplasm into compartments in an alveolatearrangement (Figures 4B and 4C). These walls are rich in cal-lose; comparable images were obtained when stained eitherdirectly with aniline blue (Figure 4B) or by the indirect immu-nofluorescence method using antibodies to 1,3-p-glucans(Figure 4C). Side views of the elongating anticlinal walls showbrightly stained bands of 1,3-p-glucans alternating with lessfluorescent regions (Figures 4B and 4C).

Concurrent with the initiation of walls in boundaries of NCDsat 5 to 6 DAP, the cytoplasm becomes more vacuolate andNCDs begin to bulge into the central vacuole (Figure 5A). Asthe nucleate columns of cytoplasm grow inward, the interphasemicrotubules become rearranged. Microtubules envelope theelongating nuclei and flare from both ends, forming a plateat the base nearest the nucellus and a crown of microtubulesin a thin layer of cytoplasm adjacent to the central vacuole (Fig-ure 5B). At 6 to 7 DAP, the cytoplasm becomes shaped intoconfigurations resembling trees. When viewed from the side(Figure 5C), the protrusions containing nuclei resemble treetrunks arising from rootlike extensions anchored in the remain-ing peripheral cytoplasm and branching into crowns thatcontact each other to form a canopy adjacent to the centralvacuole. By 7 DAP, the interaction of opposing microtubulesemanating from adjacent nuclei gives rise to phragmoplaststhat form adventitiously in the cytoplasm. These phragmoplastsare comprised of conelike subsets of microtubules on eitherside of a distinct nonfluorescent midzone (Figures 5C to 5F).The phragmoplasts are seen to be located at the interfaces

Figure 3. Syncytial Stage.

Microtubules were stained by indirect immunofluorescence; chromosomes and/or nuclei are unstained and appear black.(A) Nearly synchronous mitoses in a portion of the syncytium at 4 DAP. Interzonal phragmoplasts (various stages shown in lower portion of micro-graph) have broken down, leaving brightly fluorescent fringes (arrows) on re-forming daughter nuclei. Magnification x580.(B) Details of the mitotic apparatus in free nuclear divisions. Shown clockwise beginning at lower left are chromosomes in metaphase, anaphase,telophase, and slightly later telophase. In telophase, astral microtubules form at polar regions, and microtubules emanating from the unstainedchromosomes give rise to an interzonal array (arrow). Magnification x1060.(C) Phragmoplasts connect sister nuclei and radial microtubules interconnect non-sister nuclei. Locations of some of the nuclei (N) are shown.Magnification x530.(D) RMSs appear to organize the cytoplasm and maintain the nuclei in an evenly spaced pattern. Magnification x1460.(E) Detail of the interaction of RMSs of two adjacent nuclei. Magnification x2130.(F) Unstained zones at the perimeters of RMSs mark the location of the first walls. Magnification x1940.

Microtubules in Endosperm of Barley 1245

Figure 4. The First Anticlinal Walls.

(A) End-on view of initial walls at boundaries of syncytial NCDs asshown in Figure 3F. N indicates the position of a nucleus. Thisepifluorescence micrograph shows cell walls stained with aniline bluein UV light. Magnification xlOOO.(B) Side view of anticlinal walls separating elongated NCDs that pro-trude into the central vacuole. N indicates a nucleus. Thisepifluorescence micrograph shows cell walls stained with aniline bluein UV light. Magnification x800.(C) Same view and developmental stage as shown in (B). This portionof a section was double labeled by indirect immunofluorescence usingantibodies against 1,3-P-glucan and tubulin. The striate pattern of 1,3-(5-glucan is seen in the wall. Nuclei (one of which is labeled by theN) are shrouded by microtubules of prophase spindles. MagnificationX1100.

of microtubules emanating from adjacent nuclei in both thecanopy of cytoplasm (Figure 5E) and the peripheral floor ofcytoplasm (Figure 5F). The delineation of NCDs by radialmicrotubules in the two regions of cytoplasm continues thepattern established in the syncytium at 5 to 6 DAP when theinitial walls are deposited (Figures 3F and 4A). The free-growingwalls continue to elongate in the advancing canopy ofcytoplasm in association with the cytoplasmic phragmoplastsuntil the entire central cell is compartmentalized by closureat 8 DAP (Figure 1).

The more typical method of wall deposition in which cellplates are laid down in association with phragmoplasts follow-ing mitosis begins abruptly at 7 DAP when mitosis resumesafter a hiatus of ~2 days. By 7 DAP, nuclei in the trunklikecolumns of cytoplasm and those in the peripheral layer ofcytoplasm undergo a nearly synchronous round of mitosis fol-lowed by cytokinesis (Figures 6A to 6F). As prophase progresses,microtubules encasing the nuclei become increasingly moreorganized, and the cytoplasm becomes otherwise devoid ofmicrotubules (Figure 6A). Divisions in the columns are orientedwith spindle axes at right angles (orthogonal) to the peripheryof the embryo sac (Figures 6B to 6E). Details of mitosis aresimilar to earlier mitoses resulting in proliferation of nuclei inthe syncytium, except that now cytokinesis (Figures 6E and6F) follows immediately. Phragmoplasts develop in the inter-zones and cell plates result in periclinal walls in the columnarcells (Figure 6E). The phragmoplast/cell plates that developin association with mitotic divisions in the peripheral layer tendto be more irregularly oriented (Figure 6F). This typical formof plant cell division in which cytokinesis follows mitosis tem-porally overlaps the unusual free-growing wall formation thatinitiated compartmentalization of the central cell and continuesuntil 8 DAP (Figure 1).

Starchy Endosperm

A combination of free-growing wall formation in the advanc-ing canopy of cytoplasm and mitoses followed by cell plateformation gives rise to the starchy endosperm. Whereas cel-lularization of the central cell is completed at 8 DAP whenfree-growing walls in the advancing fronts of cytoplasm meetin the interior (closure), cell division continues to subdividethe large starchy endosperm cells for several days. Initially,the starchy endosperm consists of elongate polyhedral cellswith unusual arrays of cortical microtubules. Rather than form-ing hooplike arrays around the entire multifaceted cells, themicrotubules are parallel to each other on each facet but maybe oriented differently from those on other facets. The highlyvacuolate early starchy endosperm cells are further dividedby mitosis and cytokinesis, which takes place in a more ran-dom pattern throughout the starchy endosperm. Occasionaldivisions continue until ~14 DAP.

Figure 5. Nuclear Migration and Cytoplasmic Shaping Characterize the First Stages of Cellularization.

(A) Stereo image of a portion of the syncytium at 5 DAP showing a nucleus as it begins to bulge into the central vacuole. Magnification x3500.(B) Stereo image of a slightly later stage than shown in (A). The arrangement of nuclei (N) and associated microtubules extending into a frontof cytoplasm adjacent to the central vacuole as well as in the cytoplasm remaining at the periphery of the central cell is visible. Magnification x 1000.(C) Stereo image of treelike nucleate columns in side view showing a crown of microtubules at the top (adjacent to the central vacuole) and rootlikeprocesses extending into the peripheral region of the cytoplasm. Phragmoplasts form adventitiously in the cytoplasm where microtubules emanat-ing from adjacent nuclei (N) interact. Magnification xlOOO.(D) Enlarged area from (C) showing details of cytoplasmic phragmoplasts in the canopy of cytoplasm adjacent to the central vacuole. The opposingfanlike microtubule subsets in the phragmoplasts are especially evident in the regions marked by arrows. Magnification x1450.(E) Stereo image of a canopy of cytoplasm as seen from below showing the crowns of microtubules emanating from nuclei (one is marked withN) that appear as columns in the foreground. Magnification xlOOO.(F) Stereo image of cytoplasmic phragmoplasts in the rootlets as viewed from above. Magnification x900.

Microtubules in Endosperm of Barley 1247

Figure 6. Stereo Images Showing Cell Division in 7 DAP.

All cells are shown in side view with the central vacuole uppermost.(A) Prophase spindles in the nucleate columns. Magnification x1550.(B) Mitoses in the nucleate columns and in the remaining layer of the peripheral cytoplasm adjacent to the nucellus. Magnification x600.(C) Details of metaphase (left) and anaphase (right). Magnification x1650.(D) Details of late anaphase (left) and telophase (right). Magnification x1500.(E) Phragmoplasts originating in the interzone function in the formation of periclinal walls between daughter nuclei in the columns. Magnification xlOOO.(F) Wave of mitosis in both columns and peripheral cytoplasm showing phragmoplasts between daughter nuclei in the peripheral cytoplasm.Magnification x1200.

1248 The Plant Cell

Figure 7. Differentiation of the Aleurone Layer.

All cells are shown in side view with the central vacuole uppermost.(A) Interface of the layers of developing aleurone cells (lowermost) and starchy endosperm (uppermost). Magnification x1400.(B) Hooplike cortical microtubules in the aleurone initials. Magnification x1600.(C) At left, a PPB of microtubules in the aleurone initial predicts a periclinal division; at right, the nucleus of the same cell is shown. Magnification x2200.(D) At left, periclinal divisions in two adjacent aleurone initials show metaphase spindle and phragmoplast; at right, the metaphase chromosomesand telophase nuclei of the same two cells are shown. Magnification x2200.

Aleurone Layer

The first indication of aleurone differentiation can be detectedat ~8 DAP. Although not visible in the immunofluorescencepreparations, cytoplasm in the peripheral cell layers adjacentto the nucellus becomes denser and has a granular appear-ance as a result of the formation of numerous small vacuoles(see figures 3 and 6 in Bosnes et al., 1992). The aleurone ini-tials are initially oriented with their long axes concentric withthe nucellar surface (Figures 7A and 7B). Interphase cells ex-hibit well-ordered hooplike cortical arrays of microtubulesoriented anticlinal to the nucellar wall (Figure 7B). During aleu-rone cell development, the preprophase band (PPB) ofmicrotubules is introduced into the microtubule cycle (Figure7C). Prior to its appearance in aleurone development, thispredictive component of the cytokinetic apparatus had beennotably absent in ontogeny of the endosperm. Mitotic division

followed by phragmoplast/cell plate formation (Figure 7D) oc-curs until ~21 DAP. The completed aleurone consists of threelayers of cuboidal cells with hooplike cortical microtubulesoriented periclinally (opposite to the orientation in peripheralcells at the onset of aleurone development).

DISCUSSION

This immunofluorescence study of 4- to 21-DAP-sectioned bar-ley endosperm imaged by confocal laser scanning microscopyproduced three-dimensional information on the relationshipof microtubules to cytoplasmic organization, cell division, andwall development. Analysis of the microtubule cycle providesa new perspective on the syncytial, cellularization, and differ-entiation stages of endosperm development as defined by

Microtubules in Endosperm of Barley 1249

Bosnes et al. (1992) and Olsen et al. (1992). Whereas the mitotic apparatus is essentially the same throughout development, the pattern of cortical microtubules and the cytokinetic appara- tus differ with stages of development.

During the syncytial stage, synchronous mitoses without cytokinesis result in a rapid increase in the number of nuclei. Free nuclear division is an efficient mechanism for populat- ing the cytoplasm with numerous nuclei because divisions occur without synthesis of walls or membranes (Bennett et al., 1975). In addition, the microtubule cycle is much simplified because both components of the cytokinetic apparatus, PPBs and functional phragmoplasts, are absent. Following syncy- tia1 mitosis, microtubule systems emanating from daughter nuclei interact in the interzone, but no walls develop. In the endosperm syncytium, as well as coenocytic microsporocytes that eventually undergo simultaneous cytokinesis (reviewed by Brown and Lemmon, 1991, 1992), the organization of microtubules into the alignment typical of a phragmoplast does not necessarily bring about wall deposition. This variation in cytokinesis provides the opportunity to identify steps in the cytokinetic pathway and ultimately to understand the genes that direct and regulate cytokinesis. In both systems, RMSs emanating from the nuclei appear not only to function in spac- ing of nuclei, but also in organizing the cytoplasm into discrete units and determining the placement of walls.

Cellularization of the syncytium is a complex process that has never been fully understood. Our data, which show that cytokinesis involves a combination of the processes reported in previous studies, emphasize a prominent role of the microtubular cytoskeleton in organizing and shaping the cytoplasm into units and in the placement of walls among them. Cellularization is accomplished by two distinctly different methods of wall formation. The first, which is responsible for the initial compartmentalization of the central cell between 6 and 8 DAP, is a unique type in which walls are initiated in the absence of mitosis and, in the early stages, seem to develop in the absence of phragmoplasts. Overlapping with this un- usual type of wall formation, beginning at 7 DAP and continuing until 14 DAP, is the typical method of plant cytokinesis in which the deposition of new walls is associated with phragmoplasts that form in the interzone between daughter nuclei after mito- sis. Acting in concert, these processes bring about the controlled partitioning of the coenocytic cytoplasm.

Prior to cellularization, a unit of cytoplasm around each nu- cleus is defined by the radiating microtubules. This is taken as evidence that the endosperm syncytium is organized into NCDs similar to those recognized in other coenocytic develop- mental systems, such as simultaneous cytokinesis during microsporogenesis (reviewed by Brown and Lemmon, 1991, 1992). In simultaneous cytokinesis in moth orchids, for exam- ple, the RMSs in the four-nucleate coenocyte define the microspore domains. RMSs emanating from non-sister nuclei interact to form secondary interzonal arrays in which phrag- moplasts subsequently develop. These secondary interzonal arrays (sometimes referred to as secondary spindles), which develop in a region not previously occupied by a spindle, are

identical in structure to the primary interzonal arrays between sister nuclei. In all cases, this interzonal apparatus gradually becomes organized into a phragmoplast, which is instrumen- tal in wall deposition in simultaneous cytokinesis. In this respect, NCDs in plants differ from those documented in syn- cytial morphogenesis of certain animals and algae. In examples such as the insect Drosophila (Warn, 1986) and the alga Acetabularia (Menzel, 1986), microtubules radiating from nuclei are associated with the initial definition of NCDs but no higher order of microtubule organization is prompted at boundaries and nothing remotely resembling a phragmoplast develops. Instead, a specific organization of F-actin involved in the con- striction type of cytokinesis is localized at the boundaries of the NCDs. Because the role of F-actin in plant cytokinesis is not clear and has not been investigated in developing en- dosperm, this is an important topic for further research.

The model for the cellularization of wheat endosperm pro- posed by Fineran et al. (1982) states that all walls are formed by normal cytokinesis (i.e., guided by phragmoplasts) and do not grow freely as extensions of wall pegs protruding from the embryo sac wall as suggested by earlier workers (Mares et al., 1975, 1977; Morrison and OBrien, 1976; Morrison et al., 1978). Although our observations do not support the origin of anticlinal walls from peglike ingrowths, we did find evidence of wall formation before the appearance of phragmoplasts. van Lammeren (1988) suggested that radial microtubule systems emanating from nuclei in the syncytium may be instrumental in the development of phragmoplasts that could control for- mation of the first anticlinal walls. Although he did not show phragmoplast formation between these nuclei, and we did not observe phragmoplasts in the peripheral layer until after the initial anticlinal walls had been formed, it is clear that the RMSs are instrumental in controlling the spatial arrangement of the first formed anticlinal walls.

The development of the so called “free-growing” walls re- mains little understood. Our data on barley are in agreement with the early finding of Morrison and OBrien (1976) that these walls are rich in 1,3-B-glucans. Also, we agree that the first formed compartments are actually roofed over by cytoplasm in which periclinal walls may form, as reported by Morrison and OBrien (1976), rather than being open ended as reported by Fineran et al. (1982) and van Lammeren (1988). Phrag- moplast arrays have been reported to be associated with the tips of the free-growing walls (Mares et al., 1977; Fineran et al., 1982; van Lammeren, 1988). These arrays probably cor- respond to the cytoplasmic phragmoplasts that form in the advancing canopy of cytoplasm in barley reported herein and from earlier studies using transmission electron microscopy (see figure 8C in Bosnes et al., 1992). Dictyosomes and di- lated endoplasmic reticulum have been reported to be closely associated with the tips of such walls and have been impli- cated as a source of precursors (Bosnes et al., 1992). After the onset of phragmoplasts at ~7 DAR free-growing walls con- tinue to elongate in the advancing scaffolding of cytoplasm in association with the cytoplasmic phragmoplasts until the entire central cell is compartmentalized by closure at 8 DAP.

1250 The Plant Cell

We anticipate that further study, particularly on slow-growing mutants such as B9 (Bosnes et ai., 1992) and heavy-walled mutants (0.-A. Olsen, unpublished data), will provide more in- formation on initiation and development of the free-growing walls.

In barley endosperm, the onset of phragmoplasts occurs suddenly at -7 DAP; at this time, the cytoplasm has been rear- ranged into nucleate columns, the first anticlinal walls formed, and mitosis resumed after a 2-day hiatus. The phragmoplasts are of two types, primary and cytoplasmic or adventitious. Pri- mary phragmoplasts, which form in the interzone following mitosis, give rise to periclinal walls between daughter nuclei in the elevated columns. Cytoplasmic phragmoplasts, which form adventitiously in places other than in the equatorial re- gion of a mitotic apparatus, appear in the zones where opposing microtubule systems interact. lncreasing organization results in the typical phragmoplast configuration consisting of distinct conelike subsets of microtubules on either side of a nonfluores- cent, dark midzone in which the wall is deposited. Cytoplasmic phragmoplasts may be spatially continuous around nuclei in the canopy or initially quite discontinuous in the rootlets pos- sibly because of the numerous vacuoles in this region of the cytoplasm.

The pattern of phragmoplast development closely resembles that of the cytoplasmic phragmoplasts that form in isolated bits of coenocytic cytoplasm of Haemanthus (Bajer and Mole-Bajer, 1986) where microtubules radiating from nuclei become in- creasingly ordered, and phragmoplasts develop where microtubule systems oppose. The suborganization of micro- tubules in cytoplasmic phragmoplasts in isolated Haemanthus endosperm and in situ barley endosperm appear remarkably similar. The units of organization are like those known as microtubule converging centers where microtubules are in fan- like arrangements (Bajer and Mole-Bajer, 1982,1986; Smirnova et ai., 1992). Similar clusters of short microtubules flaring from a point have been reported at transitional stages in microtubule rearrangement in dividing cells of various plants (Schmit et ai., 1983; Gunning, 1992). It has been suggested by Bajer and Mole-Bajer (1986) that an inherent property of plant micro- tubules is responsible for this basic pattern of organization. However, results from our study show the overall organization of microtubules to be stage specific, suggesting underlying genetic regulation. Molecular studies on endosperm develop- ment will investigate the role of stage-specific gene products in control of microtubule organization and function.

The shaping of cytoplasm into nucleate columns at the on- set of cellularization may play a more prominent role in endosperm morphogenesis of barley than in wheat. Certainly there is no evidence of nuclear protrusion in figures 6 and 7 of Fineran et al. (1982); these illustrations show a phragmoplast between nuclei in the peripheral syncytium of wheat at the on- set of cellularization. To the contrary, the cytoplasm in the plane of the phragmoplast actually seems to be protruding into the central vacuole. Although we have not reinvestigated en- dosperm development in wheat, we have the impression that the syncytium at the beginning of cellularization in barley may

be thicker than in wheat. If so, crowding may be a factor in development of the protrusions of nucleate cytoplasm. In wheat, the entire syncytium is divided by walls into a single layer of compartments (Fineran et ai., 1982; van Lammeren, 1988). In barley, this stage is more variable; typically, a layer of cells is delimited between the central vacuole and a remaining layer of nucleate peripheral cytoplasm. This situation may relate to the ultimate differentiation of a multilayered aleurone in bar- ley rather than the single layer of wheat and other cereais.

The role of opposing nuclear-based microtubule systems in determining the placement of walls in endosperm development contributes basic information on the role of the cytoskeleton in the spatial control of cytoplasmic organization and wall depo- sition in plant cells. In cells with PPBs, the division site is thought to contain factors that guide and mature the leading edge of the cell plate (Mineyuki and Gunning, 1990). Although the biochemistry of the putative cytokinetic determinants is un- known, they are perhaps related to membrane proteins such as those of the ezrin-radixin-moesin family that have been shown to be specifically associated with the cleavage furrow of animal cells (Yonemura et al., 1993). It is possible that the specialized RMSs in the endosperm syncytium serve to trans- port similar cytokinetic determinants to the frontiers of the NCDs where they regulate the course of wall development.

Whereas formation of the initial walls during cellularization involves NCDs, differentiation of the peripheral layer into aleu- rone occurs by the more typical pattern characteristic of histogenesis in plant meristems (reviewed by Gunning, 1982). Interestingly, dividing cells in the aleurone meristem exhibit ali microtubule arrangements characteristic of dividing me- ristematic cells, including hooplike cortical arrays in interphase and PPBs. The original layer of cells from which the aleurone develops lacks such arrays when first delimited at 6 to 7 DA!? By 8 to 9 DAP, however, when cell division resumes, the aleu- rone initials have developed hooplike cortical microtubules resembling those of elongating cells in other plant tissues. This arrangement is strikingly different from the systems of parallel microtubules that are variously oriented on different faces of the polyhedral starchy endosperm cells to the interior. van Lammeren (1988) reported a hooplike cortical system in the developing aleurone of wheat. However, in wheat the cortical microtubules are initially periclinal in orientation, whereas in barley they are anticlinal. This difference may be reflected in development; barley aleurone becomes multilayered, whereas wheat has a single layer of aleurone cells. The cells of the aleu- rone in barley divide anticlinally to keep pace with expansion of the endosperm and periclinally to initiate additional layers of aleurone. The subaleurone cell layer at the interface of aleu- rone and starchy endosperm is intermediate in morphology (Olsen et ai., 1992). At maturity, orientation of the hooplike cor- tical microtubules is parallel to the original embryo sac wall, which is opposite to the orientation of cortical microtubules in aleurone initials at the onset of mitosis at 8 to 9 DA!?

The advent of PPBs during differentiation of aleurone has functional and evolutionary implications. The onset of PPB formation, which coincides with the development of hoop-

Microtubules in Endosperm of Barley 1251

like cortical microtubules, marks the transition to typical histo- genesis and substantiates the theory that PPBs are somehow involved in the precise control of wall placement among ad- joining cells in tissues (Mineyuki and Gunning, 1990). The meristem-like development of the aleurone could be taken as support for the evolutionary origin of endosperm as a second embryo that is developmentally suppressed and highly trans- formed.

The data reported in this study show that the cytoskeleton undergoes distinct changes in organization associated with endosperm morphogenesis. It is likely that the overall organi- zation of the cytoskeleton is under complex control involving gene products encoded by members of gene families control- ling cell cycle regulation, microtubule growth and organization, and microtubule-associated proteins that convey diverse func- tions upon the cytoskeletal framework. Preliminary studies in our laboratories indicate the feasiblility of isolating mutants in which the wild-type microtubule organization and/or func- tion is disturbed. In future studies, we will characterize developmental mutants and pursue the possibilities of using gene tagging to isolate genes controlling the major steps in cereal endosperm morphogenesis.

to suggest the red fluorescence of propidium iodide. Stereo images are portrayed as red-green anaglyphs. Hardcopy was produced by ex- porting to a film recorder. Exposures were adjusted to show the extent of the cytoplasm by a slight amount of background fluorescence. When stereo red-green anaglyphs are viewed through red (left lens)-green (right lens) stereoglasses, images should be gray-yellow on a black background and appear three dimensional. Turning the figures up- side down will reverse the perspective described in the text and figure legends.

ACKNOWLEDGMENTS

The monoclonal antibody against 1,3-P-glucans was generously sup- plied by Bruce Stone. Linda LeBlanc, Astrid Kohman, Berit Morken, and Peter Sekkelsten are thankfully acknowledged for skillful techni- cal assistance. T. Krekling is acknowledged for the micrographs in Figure 2. This work was funded, in part, by the Norwegian Research Council.

Received May 25, 1994; accepted July 18, 1994.

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DOI 10.1105/tpc.6.9.1241 1994;6;1241-1252Plant Cell

R. C. Brown, B. E. Lemmon and O. A. OlsenEndosperm Development in Barley: Microtubule Involvement in the Morphogenetic Pathway.

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