The Plant Cell, Vol. 2, 1273-1282. December 1990 O 1990 American Society of Plant Physiologists

Rearrangements of Microtubules lnvolved in Establishing Cell Division Planes Start lmmediately after DNA Synthesis and Are Completed just before Mitosis

Brian E. S. Gunning’ and Margaret Sammut Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberrs City, ACT 2601, Australia

This work concerns an aspect of spatial regulation of cell division, the development of the preprophase band (PPB) of microtubules. The PPB is significant in plant development because its position in the dividing cell indicates where the new cell wall will be inserted-an important site for control of histogenesis. We have categorized and determined the durations of stages in the development of PPBs, and have established their timing relative to the S-, G2-, and mitotic phases of the cell cycle. Roots of wheat seedlings were supplied with bromodeoxyuridine in continuous and pulse-chase treatments. Cells that were in the S-phase were identified and changes in their microtubule arrays were monitored by double immunolabeling. PPB initiation was detectable as early as the end of the S-phase as a narrowing of the preceding interphase array of microtubules. Development continued throughout G2 to a mature, narrow PPB, which existed only briefly and then eroded during the transition to the prophase mitotic spindle. The microtubule rearrangements of PPB development showed that preparation of the future site and plane of division in higher plant cells begins just after DNA replication and is completed just before mitosis.

INTRODUCTION

Many studies of the development of multicellular plant tissues have shown a virtual absence of morphogenetic migration of new cells and therefore that the placement of cell walls at the time of cytokinesis determines how cell lineages become laid out in three dimensions. Thus, it is clear that spatial regulation of cytokinesis, when the new cell wall is inserted into the framework of parental walls, is important in generating the histogenetic patterns within which developmental cell-specific and tissue-specific gene expression occurs.

Although cytokinesis occurs after mitosis, the site and plane where it takes place become distinguished before mitosis. A band of microtubules (MTs) known as the pre- prophase band (PPB) (Pickett-Heaps and Northcote, 1966a, 1966b) passes around the parental cell in the cortical cytoplasm, predicting the line where the new cell wall will fuse with the old. In some cases, it is known that the site of fusion is within a fraction of a micrometer of the midline of the PPB site (Gunning et al., 1978), even though the PPB disappears when the mitotic spindle forms, leaving its former site unmarked by any MTs during mitosis and cytokinesis (for reviews, see Gunning, 1982; Baskin and Cande, 1990).

’ To whom correspondence should be addressed.

Recent evidence indicates that the PPB site becomes specialized by insertion of factors that promote the matu- ration of the new wall. Time-lapse microscopy showed that the new wall changes from being fluid and wrinkled to being stiff and flat by about 20 min after it first attaches to the parental walls. This phase is completed if the cell plate attaches at the PPB site but not if the plate is displaced experimentally. This information suggests that the PPB MTs may direct localized deposition of wall maturing fac- tors into the parental wall or plasma membrane at the PPB site before mitosis, and that the edges of the centrifugally growing cell plate are directed to this position. The previ- ously deposited factors then pass into the new wall in the centripetal direction (Mineyuki and Gunning, 1990). To investigate this hypothesis, we have examined the timing of PPB development. The aims were to find out how the observable events among the MTs in the cell cortex relate to the phases of the nuclear cycle of cell division, and to determine the relative durations of the sequential stages, thus gaining insight into the kinetics of the events by which the cells establish their division sites and the kinetics of the observed MT reorganizations, both previously un- known quantities.

To meet these aims, it was necessary to develop a new procedure to give unambiguous evidence concerning the

1274 The Plant Cell

sequence of PPB developmental stages, which, of course,cannot be observed in vivo. In previous work, we combinedimmunofluorescence microscopy of MTs with DMA cyto-photometry to show that PPBs normally occur after DNAsynthesis in the G2 stage of the cell cycle (Mineyuki et al.,1988). That procedure was extended in the present workby marking a cohort of cells in S-phase by allowing growingroots to incorporate the thymidine analog bromodeoxyuri-dine (BrdU). Further development of this cohort was fol-lowed by sequential sampling. The marked cells wereidentified by a double-immunolabeling procedure that re-vealed the state of their MTs as they progressed throughG2 to mitosis and cytokinesis.

We have found that meristematic cells in wheat root tipsbegin to reorganize their cortical MTs at the end of theS-phase. PPB development is prolonged during G2 andleads to a distinctive mature state at prophase that con-sists of closely aggregated MTs. This phase is relativelyshort-lived and is almost immediately eroded as the spindlestarts to form.

RESULTS

Incorporation of BrdU

Figure 1 demonstrates that cells that had incorporatedBrdU into their DNA could be distinguished from those thathad not by means of anti-bromodeoxyuridine. At the sametime, the MTs could be visualized by applying anti-tubulin.It is possible, therefore, to identify cells that were in theirS-phase during exposure to the analog and to monitor thestate of the MTs in this labeled cohort.

The intensity and distribution of BrdU reactivity variedgreatly, presumably according to the stage of the S-phaseduring the exposure to the analog, and whether sufficienttime elapsed after the treatment for chromatin condensa-tion to occur. The fluorescence in the chromatin rangedfrom complete and uniform labeling to a few discretereplication sites. Treatment with 300 //M BrdU (for 10 minor longer) gave clearer labeling than lower concentrations,but 30-min exposure to 30 ^M also gave detectable incor-poration. For easy detection of labeled cells, we selected100 MM and 300 ^M as suitable concentrations of BrdU touse in continuous and pulse-labeling experiments.

Categories of Microtubule Array in Double-LabeledCells

To provide a basis for determining the developmentalhistory of PPB stages, we defined categories of MT array.To qualify for inclusion in the sample, the chromatin hadto be distinctly labeled with incorporated BrdU, the cellsintact, the MTs well preserved and labeled, and the cells

oriented so that the MT arrays on the longitudinal faceswere clearly visible. In practice, a very small proportion ofthe total cells on each coverslip met the criteria. Thepreparations also contained numerous examples of allstages of the MT cycle but with unlabeled chromatin; thesewere not counted. Labeled cells were counted by system-atically scanning all cells on the coverslips in a rasterpattern until 200 cells with labeled chromatin had beencategorized according to the state of their MTs. Replicatesamples gave closely similar data.

Cells placed in category 1 contained cortical MTs cov-ering the longitudinal faces of a cell in a standard inter-

Figure 1. File of Four Cells Isolated from a Wheat Root Tip ToShow Double-Labeling of MTs and Incorporated BrdU, and Dis-crimination of S-Phase Nuclei.

The top image shows fluorescent-labeled arrays of interphasecortical MTs, seen in profile in the cortex of every cell (betweenarrows). Incorporated BrdU is seen in the nucleus of one cell,showing that it was in the S-phase during the 30-min exposureto the analog. The bottom image is the same cell file viewed bydifferential interference contrast optics. The S-phase nucleus hasthe same appearance as the others. Scale bar = 5 ^m.

Preprophase Band Development 1275

Figure 2. S-Phase Cell with Interphase Cortical MTs (Category1).The midplane of focus (left) shows the central nucleus withuniformly labeled chromatin and unlabeled nucleoli. A few micro-tubules lie in the cytoplasm between the nucleus and the corticalarrays. The small fluorescent points in the cytoplasm may repre-sent BrdU incorporated into organelle DMA. The surface focus(right) shows numerous MTs covering the face of the cell. Scalebar = 5 Mm.

phase array. Figure 2 exemplifies this state, which wasalways the most abundant in the samples. Such cellsprovided a basis for comparing the various categories ofPPB. Category 2 consisted of wide PPBs. Figure 3 is anexample of a PPB that is more than 20 ^m wide, coveringthe whole region of the cell surface surrounding the nu-cleus. It is only slightly narrower than the interphase arrayand represents the wide extreme of category 2. Figure 4shows another wide PPB, but this example covers just aportion of the nucleus. Unlike the PPB in Figure 3, it is only2 times to 3 times wider than the narrowest type of PPB.It represents the narrow extreme of the wide PPBs, allgrouped in category 2. Categories 3 and 4 contained thenarrowest PPBs. Figure 5 shows a narrow but fibrillar PPB(category 3). In category 3, the constituent MTs (or bundlesof MTs) were visible; in category 4, the PPB was almostuniformly fluorescent, without obvious fibrillar appearance.Figure 6 shows a narrow PPB (category 4) in which indi-vidual MTs cannot be discerned. The remaining categoriesinvolved stages of spindle or phragmoplast development.Cells with eroded, visibly fibrillar PPBs in combination withprespindle MTs aligned on the nuclear surface in the pole-to-pole axis were grouped in category 5 (not illustrated,although a few MTs can be seen on the surface of thenucleus in Figure 4). Cells in metaphase, anaphase, andtelophase were grouped as mitotic stages, using the termloosely, as cells in prophase were excluded (not shown).Cells that had reached the stage of cytokinesis werealso recorded. Figure 7 shows a cell that had passed

through G2 and mitosis to a late stage of phragmoplastdevelopment.

In addition to the stages described above, cells that hadrecently completed cytokinesis and were reinstating theircortical MT arrays were identified in samples taken 3.5 hrto 4 hr after a pulse-label period in BrdU. Thus, BrdUincorporation does not necessarily block completion of oneMT cycle and restarting of the next. However, with longerchases, it was not possible to distinguish with certaintynew progeny of mitosis and cytokinesis in BrdU-labeledcells from cells that might have passed through S and thenstayed in the interphase (category 1) state throughout thechase period. For this reason, chase treatments longerthan 4 hr were not employed: in any case, all of thecategories of interest had appeared in less time than this.

Microtubules in S-Phase Cells

To pinpoint the onset of PPB development with greatestaccuracy, we tried giving very short exposures to theanalog. The shortest treatment was 10 min (300 //M). Inroot tips sampled at the end of the pulse in that experiment,1 % of cells with labeled chromatin had wide PPBs (cate-gory 2). In four other experiments using 30-min pulses, theaverage count was 0.6% ± 0.34 in samples taken at the

Figure 3. Cell with Labeled Chromatin and a Wide PPB, Close tothe Widest Extreme within Category 2.

The two ends of the cell are just outside the left and right limitsof the micrograph so the PPB occupies about two-thirds of theface of the cell. The midplane of focus (top micrograph) showsthe MTs of the PPB in profile, concentrated around the nucleus.The surface focus (bottom micrograph) shows that the MTs aredisordered or lost at the extremities of the cell. Many stragglefrom the poles of the nucleus. This is an early stage of PPBdevelopment. Scale bar = 5 ^m.

1276 The Plant Cell

Figure 4. BrdU-Labeled Cell with a PPB at the Narrow Extremeof Category 2.

These fluorescence (top) and differential interference contrast(bottom) views depict a cell with a PPB that is about 2 times to 3times wider than its final state. In the top picture, the plane offocus is just within the nucleus to show the BrdU-labeled chro-matin together with oblique and profile views of the PPB MTs (atdouble arrows). The fluorescence of the chromatin is patchy,suggesting that the cell may have been exposed to BrdU late inS so that only late-replicating DMA has incorporated the label.The bottom picture shows that at this stage of PPB developmentthe chromatin has not yet condensed and the nucleolus (openarrow) is still present. Scale bar = 5 ^m.

end of the pulse. No other categories of PPB were foundin the labeled population. All other cells were in category1 (interphase arrays).

The 10-min and 30-min BrdU pulses could have beenlocated anywhere within the S-phase, which in wheat roottip cells lasts 4.6 hr at 20°C (Francis et al., 1985). Thefinding that more than 99% of S-phase cells retain the

interphase MT arrangement showed, therefore, that thePPB is not initiated earlier than the end of the S-phase. Itis not justifiable to conclude that they begin to form whenthe S-phase is 99% complete because the length of thepulse relative to the length of S does not admit such highresolution. In some cases, the pulse would have spannedthe end of the S-phase and the beginning of G2, so it ispossible that the few BrdU-labeled cells with category 2PPBs had completed DMA synthesis during the treatmentand had then begun to rearrange their MTs in the firstminutes of G2 while still in BrdU solution. We investigatedthese possibilities by treating roots for longer periods andin pulse-chase treatments.

The Sequence of Preprophase Band Developmentduring Continuous Exposure to BrdU

All PPBs seen in BrdU-labeled cells at the end of 10-mintreatments were in category 2. It follows that these widePPBs were indeed early stages of development. Figure 8shows that all four categories of PPB accumulated during2 hr of continuous exposure to BrdU. Wide PPBs (category2) were the first to increase in frequency in the labeledcohort of cells. The first narrow, fibrillar PPBs (category 3,

Figure 5. Two Planes of Focus of a Cell with a Narrow but StillFibrillar PPB (Category 3).

In this cell, the BrdU labeling is confined to discrete patches ofchromatin, perhaps indicating that it was labeled very late in S.Although the PPB is condensed to about 4 ^m wide, the surfacefocus still shows a fibrillar texture. A few MTs remain outside thePPB, at random on the nuclear surface (right image) and clusteredaround the nuclear poles (left image). Scale bar = 5 nm.

Preprophase Band Development 1277

Figure 6. Mature Stage of PPB Development (Category 4).Three views of a cell with condensed chromatin (examples atwhite arrows in lower picture) that is labeled with BrdU (top andmiddle pictures). The mature PPB, 3 ^m wide, is seen in profile inthe midplane of focus in the top picture and in surface view in the

labeled "narrow" in Figure 8) appeared after 30 min andthe first narrow, nonfibrillar PPBs (category 4, labeled"mature") after 90 min. PPB-spindle transitions (category5) were also first seen at 90 min. Categories 2 to 5increased in frequency as more and more cells passedthrough the S-G2 transition and entered the successivestages of PPB development. All of the remaining cells inthe labeled cohort were in category 1. They declined infrequency as the other categories accumulated and werenot included in the graph.

The results established the overall sequence of PPBdevelopment but did not indicate the extent to which theBrdU-labeled cell population behaves synchronously. Forinstance, it is possible that the accumulation of cells withcategory 2, wide PPBs, occurred because all of the cellsthat leave S (provided that they have been committed todivide rather than to arrest in G2) modify their interphasearrays at the end of the S-phase, or because some cellsdo so at intervals thereafter in G2. It is known that somecells in onion root tips arrest in G2 without undergoingPPB development (Mineyuki et al., 1988). Nevertheless, itcan be concluded that the developmental sequence forPPBs begins, for at least some cells, very close to the endof the S-phase and that it consists of progressive narrow-ing of the band of MTs until they are so crowded that thevisualization method no longer resolves a fibrillar compo-sition. The intervals between the successive plots indi-cated that on average the narrowing process is prolonged,whereas the category 4 state is relatively short-lived andgives way quickly to PPB-spindle transitions. Assumingthat this transition signals that the M-phase is imminent, itfollows that for at least some cells, PPB narrowing andmaturation occupy essentially the whole of G2. Figure 6shows that by the time mature, category 4 PPBs havedeveloped, the chromatin has condensed and the nucleoliare beginning to disperse, features of prophase not seenearlier in PPB development.

The Sequence of Preprophase Band Development inPulse-Chase Treatments with BrdU

In pulse-chase treatments, the intensity of labeling usuallyincreased with increasing chase periods up to about 90min, probably because the root accumulated a pool ofBrdU during the pulse, thus providing for continued incor-poration into DNA. Figures 9 and 10 show data fromexperiments that employed 100 /^M and 300 nM BrdU,

middle picture. Individual MTs (or bundles of MTs) are not resolv-able in the PPB. A few MTs are lined up in the pole-to-pole axison the nuclear surface (middle picture). The nucleoli are much lessclear than in earlier stages but have not yet dispersed (largearrows in bottom picture) and the nuclear envelope is still intact(bottom picture). Scale bar = 5 ̂ m.

1278 The Plant Cell

Figure 7. Cell That Completed Cytokinesis 3.5 hr after a 30-minPulse Label in BrdU.This cell progressed from its S-phase through G2 and mitosis toa late stage of cytokinesis with peripheral phragmoplast MTs(arrows) despite having taken BrdU into its chromatin. Nucleoli(bottom picture) are re-forming in the daughter nuclei, which havepatchily labeled chromatin (top picture). The cell plate is not visibleand probably has been digested by the treatment used to free thecells from the root tip. Scale bar = 5 ^m.

respectively, in 30-min pulses, followed by a range of chasetimes. The chase periods were chosen to encompassstages of PPB development up to the onset of mitosis andcytokinesis in BrdU-labeled cells. The first BrdU-labeledcells reached the stage of cytokinesis after 2.5 hr (Figure7). By 4 hr, the numbers of cells in cytokinesis matched

those of categories 2 and 3 and mitotic stages at 11 % to14%, outnumbering category 4 by twofold (data notshown).

PPB narrowing was again seen to be prolonged andgradual, preceding formation of the mature, nonfibrillarPPB, which was followed closely by PPB-spindle transi-tions. Differences between the two concentrations of BrdUwere few. The plateau in the frequency of wide PPBs andthe steep rise in numbers of narrow PPBs when 300 nMwas used (Figure 10, cf. Figure 9) may indicate that thehigher concentration inhibited maturation to category 4 insome cells. Roots on seedlings treated with 300 juM BrdUas in the sampled material continued to grow, and mitoticand cytokinetic stages continued to be reached in longerchases so inhibitory effects were at most partial and didnot affect the main conclusions regarding the develop-mental sequence and the relative timing of the stages.

DISCUSSION

We confirmed that PPBs are features of the G2 period ofthe cell cycle (Mineyuki et al., 1988), and showed further

30 60 90Minutes in bromodeoxyuridine

Figure 8. Time Course of Accumulation of Categories of PPBduring Continuous BrdU Treatment.

The points are percentages of the total sample of BrdU-labeledcells. Cells not accounted for in the plots had interphase corticalarrays (category 1), as illustrated in Figures 1 and 2.

Preprophase Band Development 1279

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.- 2 12- 2 2 1 6 m

o

.- 12- e, o

2 10- d e, e, 3

I

Minutes after 30 min broniodeoxyuritline pulse

Figure 9. Time Course of Accumulation of Categories of PPB and Subsequent Stages of the Microtubule Cycle after a 30-min Treatment with 1 O0 pm BrdU.

that they start to develop at the end of the S-phase in at least some cells, although it was not excluded that in other cells there could be a later start. Development progressed through G2 by narrowing of the PPB until the constituent MTs were in an approximately 3-fim-wide girdle. As judged by what happens in the population, this process was prolonged and gradual. It was followed by maturation, defined here as the process by which the MTs of the band become so closely aggregated that immunofluorescence microscopy cannot resolve them. The minimum time to reach this mature state was 90 min to 120 min from PPB initiation. This stage was (again, on a population basis) short-lived, was accompanied by condensation of the chro- matin, and was closely followed by the appearance of spindle precursor MTs on the nuclear surface. The possi- bility that mature PPBs (category 4) were infrequent be- cause some cells omitted this highly aggregated nonfibrillar stage was considered remote because of the close kinetic parallel between categories 4 and 5 and because all early stages of PPB-spindle transition (with not many pole-to- pole MTs) had nonfibrillar PPBs. Only later, when there were many pole-to-pole MTs, did the PPBs become visibly fibrillar again through erosion of their MTs.

Thus, our kinetic analysis gives a solid basis to the postulated sequence from wide, diffuse PPBs to narrow, dense PPBs first proposed by Wick and Duniec (1983, 1984) and elaborated more recently by Wang et al. (1 989) and Mineyuki et al. (1989). It also puts on a firm time scale

other developmental studies such as our own correlations of actin redistributions with stages of PPB formation and decline, which at the time were merely putative (McCurdy et al., 1988; McCurdy and Gunning, 1990). To our knowl- edge, the only comparable investigation concerns proto- nema apical cells in the fern Adiantum, where a diffuse band beside the prophase nucleus forms during G2, per- haps starting in late S (Wada et al., 1980).

The conclusion that in wheat root tip cells PPB devel- opment can be seen first at or close to the end of the S-phase is not quite the same as that obtained for onion root tips using 30-min pulses of 3H-thymidine and autora- diography to identify S-phase cells (Mineyuki et al., 1988). In Mineyuki et al. (1988), two out of 11 such cells had detectable PPBs; in this study, only 0.6% of a sample nearly 100-fold larger had early PPBs. In both studies, the frequency of cells with PPBs (and subsequent stages) rose during the chase periods, but the rises were much steeper in onion and reached 83% to 100% of labeled cells in some samples. (PPB stages were not determined.) In wheat, the highest frequency recorded was 59% after a 4-hr chase period, although there was no sign that a peak had been reached. (With a 4.6-hr S-phase, a peak would not be expected within that period.) Comparing the 3H-thymidine and BrdU procedures, it appears that PPB

f I

narrow I

I I

I

3 v) - e 8 4-

€9

% O

2- L

0 30 60 90 120 150 180 210 Minutes after 30 min bromodeoxyuridine pulse

Figure 10. Time Course of Accumulation of Categories of PPB and Subsequent Stages of the Microtubule Cycle after a 30-min Treatment with 300 pm BrdU.

1280 The Plant Cell

development may start earlier in onion. To check this point, we gave onion seedlings a 30-min BrdU treatment and found that 2% of labeled cells had early stage PPBs at the end of this period. Although this fraction was somewhat larger than that found in wheat, it was not large enough to confirm the results obtained earlier by applying 3H- thymidine to onion. The 3H-thymidine results may, therefore, have been misleading because so few clearly labeled cells were found as a result of the severe technical problems of combining tubulin immunofluorescence with microautoradiography.

Some authors have queried the use of the term prepro- phase to describe the PPB, pointing out that in their material the chromatin has a prophase appearance when the band is present (e.g., Roberts et al., 1985; Hogan, 1988; Meijer and Simmonds, 1988). The term prophase band would indeed be appropriate for the morphologically distinct, close-packed, mature state (category 4), which we have shown is short-lived, correlated with condensed chromatin, and is eroded when the extranuclear pole-pole spindle MTs are laid down. By contrast, the preparations, from S through G2, are truly preprophase. Because the band is present, albeit in an immature state, for such a prolonged period, we recommend that the term prepro- phase should be retained.

One question arising from the observation that PPB development occurs throughout G2 concerns integration of the cytoplasmic events of preparation for cytokinesis with the nuclear events of the S-phase and mitosis. Does PPB development require a certain time and does it trigger the transition to the spindle when it has been completed? Conversely, is the timing of PPB development controlled by other elements of the cell cycle so that it always starts at the end of S and proceeds rapidly or slowly according to whether G2 is short or prolonged? The double-labeling procedure that we have used should enable this question to be answered.

For root meristem cells, we now know (1) that the first visible signs of preparations for cytokinesis (PPB devel- opment) occur later than the onset of the nuclear events (DNA replication); (2) that PPBs can occasionally develop even if DNA replication is inhibited, showing that the two control paths are separable, although they normally occur in fixed sequence (Mineyuki et al., 1988); (3) that PPB development is a gradual progression to a narrow band that exists briefly at prophase and is well known from other work (see Gunning, 1982) to predict precisely where cy- tokinesis will occur; (4) that actin is redistributed from the cell interior to the cell cortex while the PPB is narrowing, but not with the same precisely defined and located end product as in the case of the PPB (McCurdy and Gunning, 1990); and (5) that PPB narrowing is sensitive to cyto- chalasin D (Mineyuki and Palevitz, 1989).

It is well established that the PPB marks the division site of the cell, where the cell plate will attach to the

parenta1 walls, irrespective of whether the division is trans- verse, longitudinal, oblique, symmetrical, or asymmetrical (reviewed by Gunning, 1982; Baskin and Cande, 1990). Venverloo (1 990) has shown that wound-induced divisions can be reoriented into a new plane by rewounding, pro- vided that the second wound is imposed within a limited time period. The plane of division later becomes irreversibly determined and second wounds then have no reorienting effect. Taken together, our observations on the sequence and timing of MT rearrangements and the experiments of Venverloo (1 990) on induced alteration of division planes during G2 indicate that the division site is established at the culmination of gradual and initially plastic events that take place in the cell cortex throughout G2.

Evidence concerning the functional role of the PPB site during cytokinesis points to two phases of activity (Mine- yuki and Gunning, 1990). In the first phase, factors that function in maturing the new cell plate, which could be receptors for Golgi vesicle fusion or enzymes of wall metabolism, are postulated to be deposited in the plasma membrane or cell wall at the division site, most probably under the directional guidance of the PPB MTs. The kinetic data presented here offer two possibilities for how this could happen. The narrowing of the interphase array of MTs throughout G2 could congregate membrane proteins from a dispersed state, where they function in general wall biosynthesis, into the approximately 3-pm-wide girdle of the PPB site. They remain there until the arrival of the leading edge of the cell plate as it grows centrifugally during cytokinesis. The second possibility is that the cell plate maturation factors are secreted into place under the guidance of the MTs during the brief time when the PPB is mature, that is, when it is placed most accurately in the cell. The second functional phase of the PPB site is seen at cytokinesis, rather than when the PPB itself is present. The cell plate grows centrifugally from its origin at the spindle equator until it reaches the vicinity of the PPB site. It then comes under the influence of the site and grows toward the midline of the zone that was defined, during G2, by the developmental processes whose sequence and kinetics have been the subject of the present investigation.

METHODS

Wheat (Triticum aestivum cv Kite) seeds were germinated on moist filter paper and grown for 2 days to 3 days in inclined dishes at 25'C in darkness and used when the roots were 1 cm to 2 cm long. This variety of wheat was also used in other recent studies of cytoskeletal rearrangements at division sites (Marc and Gun- ning, 1988; McCurdy et al., 1988; McCurdy and Gunning, 1990).

Some experiments involved continuous exposure of intact seedlings to BrdU (Sigma) dissolved in water at concentrations of 30 pM, 1 O0 pM, or 300pM. In most, however, pulse-chase regimes were used. In such cases, the seedlings were treated in BrdU

Preprophase Band Development 1281

solutions for 10 min or 30 min and then rinsed (three times in distilled water) and returned to water for periods up to 4 hr. All treatments were conducted in darkness at 25'C.

Portions of root tip 1 mm long were excised and fixed for 1 hr at 25OC in 4% paraformaldehyde in PM5E buffer (microtubule- stabilizing buffer consisting of 50 mM Pipes, 2 mM magnesium sulfate, and 5 mM EGTA at pH 6.8) and rinsed (3 x 5 min) in PM5E buffer. The excised tips were treated with 1% cellulysin (Behring Diagnostics) in PM5E buffer for 40 min at 25OC, rinsed again, and squashed between coverslips that had previously been coated with poly-L-lysine (W, 540,000, Sigma).

Successful double-labeling with monoclonal anti-BrdU (Becton Dickinson catalogue No. 7580, Lane Cove, NSW, Australia) and monoclonal anti-p-tubulin (Amersham, Sydney, Australia) was ac- complished by a protocol in which the anti-BrdU, anti-p-tubulin, and fluorescein isothiocyanate (FITC)-conjugated second antibody followed each other in three stages (with intervening rinses). Labeling of the incorporated BrdU was inconsistent, however, and a longer protocol in which the same second antibody followed each primary antibody in a total of four stages was preferable. In neither protocol was it necessary to use different fluorochromes to discriminate between bound anti-BrdU and anti-0-tubulin be- cause the two primary antibodies were directed against structur- ally and spatially discrete targets (chromatin and MTs). In both protocols, the fixed cells on the coverslips were first treated with 1.5 N HCI to render incorporated BrdU accessible to its antibody (Levi et al., 1987). Preliminary work established that a 40-min irrigation with HCI provided a usable compromise: shorter periods reduced labeling of incorporated BrdU and longer periods reduced labeling of MTs with anti-p-tubulin. Anti-BrdU was used at 1:5 dilution and anti-8-tubulin at 1 :500; the labeled second antibody was FITC-conjugated sheep anti-mouse lg antibody (Silenus Lab- oratories Pty. Ltd., Dandenong, Australia) diluted 1 :30. All anti- bodies were diluted in buffered saline (16 mM Na2HP04, 4 mM NaH2P04, 150 mM NaCI, pH 7.2) containing 1% bovine serum albumin (fraction V, Sigma) and 0.02% sodium azide, and all treatment periods were 40 min at 37OC, always followed by 3 x 5-min rinses in PBS. At the end of the antibody treatments, the coverslips were rinsed in distilled water, drained, and mounted in glycerol-polyvinyl alcohol (mowiol, Hoechst) medium (Osborn and Weber, 1982) containing 0.1 YO aqueous p-phenylenediamine (Sigma) (Valnes and Brandtzaeg, 1985).

Observations were made using a Nikon Optiphot microscope with differential interference contrast and epifluorescence optics with a standard FlTC filter set. lmages obtained from a SIT television camera (Dage MTI, Michigan City, IN) were digitized and recorded using an Image-1 image processing attachment (Universal lmaging Corporation, West Chester, PA)

ACKNOWLEDGMENT

We thank Professor Tom Rost, University of California, Davis, for drawing our attention to the availability of anti-BrdU.

Received July 3, 1990; accepted October 1 1, 1990.

REFERENCES

Baskin, T.I., and Cande, W.Z. (1990). The structure and function of the mitotic spindle in flowering plants. Annu. Rev. Plant Physiol. Plant. MOI. Biol. 41, 277-315.

Francis, D., Kidd, A.D., and Bennett, M.D. (1985). DNA replica- tion in relation to DNA C values. In The Cell Division Cycle in Plants, J.A. Bryant and D. Francis, eds (Cambridge: Cambridge University Press), pp. 199-21 5.

Gunning, B.E.S. (1982). The cytokinetic apparatus: Its develop- ment and spatial regulation. In The Cytoskeleton in Plant Growth and Development, C.W. Lloyd, ed (New York: Academic Press), pp. 229-292.

Gunning, B.E.S., Hardham, A.R., and Hughes, J.E. (1978). Pre- prophase bands of microtubules in all categories of formative and proliferative cell division in Azolla roots. Planta 143,

Hogan, C.J. (1988). Preprophase bands in a suspension culture of the monocot Spartina pectinata. Exp. Cell Fies. 175,

Levi, M., Spanroli, E., Sgorbati, S., and Chiatante, D. (1987). Rapid immunofluorescent determination of cells in the S phase in pea root meristems: An alternative to autoradiography. Phys- iol. Plant. 71, 68-72.

Marc, J., and Gunning, B.E.S. (1 988). Monoclonal antibodies to a fern spermatozoid detect nove1 components of the mitotic and cytokinetic apparatus in higher plant cells. Protoplasma

McCurdy, D.W., and Gunning, B.E.S. (1 990). Reorganization of cortical actin microfilaments and microtubules in wheat root-tip cells: A double label immunofluorescence study . Cell Motil. Cytoskeleton 15, 76-87.

McCurdy, D.W., Sammut, M., and Gunning, B.E.S. (1988). Im- munofluorescent visualization of arrays of transverse cortical actin microfilaments in wheat root-tip cells. Protoplasma 147,

Meijer, E.G.M., and Simmonds, D.H. (1 988). Microtubule orga- nization during the development of the mitotic apparatus in cultured mesophyll protoplasts of higher plants-An immunoflu- orescence microscopic study. Physiol. Plant. 74, 225-232.

Mineyuki, Y., and Gunning, B.E.S. (1990). A role for preprophase bands of microtubules in maturation of new walls, and a general proposal on the function of preprophase band sites in cell division in higher plants. J. Cell Sci. 97, 527-537.

Mineyuki, Y., and Palevitz, 8. A. (1989). Cytochalasin inhibits microtubule bundling in preprophase cells of Allium epidermis. Cell Struct. Funct. 14, 887.

Mineyuki, Y., Wick, S.M., and Gunning, B.E.S. (1988). Prepro- phase bands of microtubules and the cell cycle: Kinetics and experimental uncoupling of their formation from the nuclear cycle in onion root-tip cells. Planta 174, 51 8-526.

Mineyuki, Y., Marc, J., and Palevitz, B. A. (1989). Development of the preprophase band from random cytoplasmic microtubules in guard mother cells of Allium cepa L. Planta 178, 291-296.

Osborn, M., and Weber, K. (1982). lmmunofluorescence and immunocytochemical procedures with affinity purified antibod- ies: Tubulin-containing structures. In Methods in Cell Biology,

145-160.

21 6-222.

142,15-24.

204-206.

1282 The Plant Cell

Vol. 24, The Cytoskeleton, part A, L. Wilson, ed (New York: Academic Press), pp. 97-132.

Pickett-Heaps, J.D., and Northcote, D.H. (1 966a). Organization of microtubules and endoplasmic reticulum during mitosis and cytokinesis in wheat meristems. J. Cell Sci. 1, 109-120.

Pickett-Heaps, J.D., and Northcote, D.H. (1 966b). Cell division in the formation of the stomatal complex of the young leaves of wheat. J. Cell Sci. 1, 121-128.

Roberts, K., Burgess, J., Roberts, I., and Linstead, P. (1985). Microtubule rearrangements during plant cell growth and de- velopment: An immunofluorescence study. In Botanical Micros- copy 1985, A.W. Robards, ed (Oxford: Oxford University Press),

Valnes, K., and Brandtzaeg, P. (1985). Retardation of immuno- fluorescence fading during microscopy. J. Histochem. Cyto- chem. 33,755-761.

Venverloo, C.J. (1990). Regulation of the plane of cell division in vacuolated cells. ( I . Wound-induced changes. Protoplasma 155, 85-94.

pp. 263-283.

Wada, M., Mineyuki, Y., Kadota, A., and Furuya, M. (1 980). The changes of nuclear position and distribution of circumferentially aligned cortical microtubules during the progression of cell cycle in Adianfum protonemata. Bot. Mag. 93, 237-245.

Wang, H., Cutler, A.J., and Fowke, L.C. (1989). High frequencies of preprophase bands in soybean protoplast cultures. J. Cell Sci. 92, 575-580.

Wick, S.M., and Duniec, J. (1 983). lmmunofluorescence micros- copy of tubulin and microtubule arrays in plant cells. 1. Prepro- phase band development and concomitant appearance of nu- clear envelope-associated tubulin. J. Cell Biol. 97, 235-243.

Wick, S.M., and Duniec, J. (1 984). lmmunofluorescence micros- copy of tubulin and microtubule arrays in plant cells. I I . Transi- tion between the pre-prophase band and the mitotic spindle. Protoplasma 122,45-55.

DOI 10.1105/tpc.2.12.1273 1990;2;1273-1282Plant Cell

BES. Gunning and M. SammutImmediately after DNA Synthesis and Are Completed just before Mitosis.

Rearrangements of Microtubules Involved in Establishing Cell Division Planes Start

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