High frequencies of preprophase bands in soybean protoplast cultures

HONG WANG1'2, ADRIAN J. CUTLER2 and LARRY C. FOWKE1'*

department of Biolog)*, University of Saskatchewan, Saskatoon, Saskatchewan S7N 0W0, CanadazPlant Biotechnology Institute, National Research Council, Saskatoon S7N 0W9, Canada

•Author for correspondence

Summary

Protoplasts derived from a non- regenerable soy-bean cell line provide an excellent experimentalsystem for studying plant preprophase bands(PPBs). Cultured protoplasts developed PPBs inhigh frequencies, permitting a detailed analysis ofPPB development. From observations of thousandsof PPBs, six distinct developmental stages wereidentified. This classification should prove useful inrecognizing developmental stages of PPBs and incomparing results among different tissues andspecies. Perinuclear fluorescence appeared whenPPBs were well developed. It consisted of micro-tubule strands radiating from the nuclear surface atits early stages and more extensive arrays on thesurface at later stages.

All protoplast cultures from 0-5 day to 5 days hada PPB index (ratio between number of PPBs andnumber of phragmoplasts) greater than unity,suggesting that in this cell line a high frequency ofPPBs is apparently not related to the potential fororganized growth (e.g. embryogenesis). Differentmethods of quantifying the occurrence of PPBswere evaluated. The PPB value (ratio between % ofcells with PPB and the mitotic index) was comparedwith the PPB index and results indicate that it is auseful new parameter for studies of the distributionof PPBs in plant cells.

Key words: immunofluorescence microscopy, preprophaseband microtubules, protoplast culture, soybean.

Introduction

The preprophase band (PPB), a bundle of microtubules(MTs) girdling the cell cortex around the nucleus prior toprophase, is believed to predict the site where a new cellplate will fuse with the parental cell wall (Gunning,1982). Since their first discovery in wheat (Pickett-Heaps& Northcote, 1966a,b), PPBs have been reported intissues of a variety of higher plant species (Gunning,1982; Gunning & Hardham, 1982). As they were initiallynot observed in tissues exhibiting unorganized growth,e.g. endosperm cells (De Mey et al. 1982; Schmit et al.1983), cultured cells and protoplasts (Fowke et al. 1974),PPBs were thought to be characteristic of cells inorganized plant tissues. In such tissues PPBs wouldpredict a precise division site, leading to cell polarity.

Cultured plant cells and protoplasts are unique exper-imental tools of increasing importance for both funda-mental biology and biotechnology. Unlike cells in planttissues, cultured cells are more easily controlled inproliferation and differentiation and usually are morehomogeneous when differentiation does not occur. Re-cently, due to the use of indirect immunofluorescencemicroscopy (see Lloyd, 1987), PPBs have been revealedin cultured cells of Vicia hajastana (Simmonds & Setter-Journal of Cell Science 92, 575-580 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

field, 1983; Simmonds, 1986), Zinnia elegans (Falconer& Seagull, 1985), tobacco (Gorst et al. 1986), carrot(Gorst et al. 1986; Doonan et al. 1987) and Spartinapectinata (Hogan, 1988). Thus PPBs are not restricted tocells with organized growth. However, most of thesestudies did not provide quantitative data on the occur-rence of PPBs. In one case where quantitative studieswere done using cells from suspension cultures and roottips of tobacco and carrot (Gorst et al. 1986), it wassuggested that cells having the potential for morphogen-esis had a higher PPB index (the ratio between number ofPPBs and number of phragmoplasts). The PPB indexmight thus have significant applications in plant biotech-nology; for example, by facilitating the selection ofregenerable cell lines. However, it is not clear to whatdegree this idea holds true and whether it will proveuseful. Therefore the frequency of PPBs should bedetermined for a variety of cell cultures with establishedstatus of regenerability to assess the validity of thisintriguing idea.

The present study: (1) reports high frequencies ofPPBs in protoplast cultures of a non-regenerable soybeancell line; (2) provides a detailed description of theorganization and development of PPBs in these cells; and(3) proposes an alternative index for quantifying PPBs.

575

Fig. 1. Protoplasts from soybean suspension cell line SB-1. X 100. A. Freshly isolated protoplasts. B. Cell divisions after 2 daysof culture.

Materials and methods

Cell lineThe soybean (Glycine max (L.) Merr.) cell line (SB-1) wasestablished in 1966 by Gamborg. It consists of small cellclusters capable of rapid cell division. Shoots or embryos havenever been induced from this culture despite numerous at-tempts (F. Constabel, K. N. Kao & J. Shyluk, Plant Biotech-nology Institute, National Research Council of Canada, per-sonal communications). Thus the line is considered non-regenerable in a practical sense. Cells were cultured in 1-B5medium (Gamborg, 1982) as described by Saleem & Cutler(1986).

Protoplast isolation and cultureTwo days after subculture cells were resuspended in enzymesolution consisting of 0-6% cellulase Onozuka R-10 (Kane-matsu-Gosha Ltd, CA, USA), 0-2% driselase (PlenumScientific Research Inc., Hackensack, NJ, USA), 0-2% hemi-cellulase (Rohm & Haas Co. Canada Ltd, West Hill, Ontario,Canada) and 0 2 % pectinase (Terochem Laboratories Ltd,Edmonton, Alberta, Canada) in 0-55 M-sorbitol with pHadjusted to 5-8 and incubated in darkness on a shaker (approx.50revsmin~') for 12-14 h. The resulting protoplasts werefiltered through a 48//m nylon mesh. After washing twice in0-55 M-sorbitol solution (pH 5-8), protoplasts were resuspendedin the protoplast culture medium (Kao, 1982) and cultured at adensity of 4x 10s ml"1.

Immunofluorescence microscopyFreshly isolated protoplasts were fixed in 3-7% formaldehydein MT stabili2ing buffer (MTSB) (100mM-l,4-piperazine-diethylsulphonic acid (Pipes), 1 mM-MgSO4 and 2mM-ethyl-eneglycol-bis-(aminoethylether)-Ar

f/Vf/V',Ar'-tetraacetic acid(EGTA), pH6-9) for 8h. Protoplast cultures (>0-S day) werefixed in the above fixative containing 0 1 % Triton X-100 for4h. The fixed protoplasts and cells were attatched to coverslipspreviously coated with poly-L-lysine (Mr 564 000, Sigma, StLouis, MO, USA) and extracted in 1 % Triton X-100 in MTSBfor 1 h. They were then treated in 0-1 % NaBH+ in phosphate-buffered saline (PBS) (0-14M-NaCl, 2-7mM-KCl, 8-0 mM-Na2HPO4, l-5mM-KH2PO4, pH70) for 6min and rinsedthree times in PBS. A 200 [A sample of monoclonal rat anti-yeasttubulin antibody (Dimension Laboratories, Mississauga,Ontario, Canada), diluted 1:300 in PBS, was layered on eachcoverslip and incubated for 45 min, and then rinsed three timesin PBS. The coverslip was then treated with fluorescein-

conjugated rabbit anti-rat immunoglobulin antibody (ICNImmunoBiologicals, Lisle, IL, USA) diluted 1:150 (v/v) inPBS. Nuclei were stained with Hoechst 33258 (lOjugml"1).Coverslips were mounted on glass slides in PBS containing 2%w-propyl gallate (Sigma) and 50% glycerol. Preparations wereexamined using a Zeiss Universal epifluorescence microscopewith a 63 X lens (NA = 1-40) and photographs were taken onIlford XP1-400 film.

Quantification of PPBsThe following parameters were determined to provide quanti-tative data on PPBs. (1) PPB index, the ratio between thenumber of PPBs and the number of phragmoplasts in a sampleof cells (Gorst et al. 1986); (2) % of PPB cells, the percentage ofcells with a PPB in a sample of cells; (3) mitotic index (% ofcells from prophase to telophase); and (4) PPB value, the ratiobetween (2) and (3) (i.e. between the % of PPB cells and mitoticindex). To obtain the PPB index, PPBs and phragmoplastswere counted to a sum of 100 in each of five or more samples andthe ratio was individually calculated. The number of cellsscreened for each treatment = (no. of PPB cells counted)/(% ofPPB cells). For the PPB value, 500 cells were counted for the %of PPB cells and mitotic index in each of three to five samples.The variation of estimates generated from both methods wasevaluated by the coefficient of variation, c.v. = o~,,-\Jx, where .vis the mean for one treatment and an-\ is the standard deviationof the mean.

Results

Protoplast cultures and MT stainingProtoplasts prepared from this cell line were quiteuniform and most of them contained one nucleus(Fig. 1A). Cells formed from the protoplasts dividedwithout a long lag phase. After 24h in culture 28-8% ofthe surviving protoplasts had divided; most divisionsappeared normal (Fig. IB).

MTs in all phases of the cell cycle were well stained andnon-specific background fluorescence was low. Omittingthe primary antibody or the secondary antibody, orreplacing the primary antibody with normal rat anti-serum, produced little general fluorescence.

PPBs during ptvtoplast cultureFreshly isolated protoplasts contained randomlyarranged MTs (Fig. 2A) and no PPBs were observed.

576 H. Wang et al.

Fig. 2. Occurrence of preprophase bands (PPBs) during culture of soybean protoplasts. A-G. X980; H, X680. A. Freshlyisolated protoplast showing randomly arranged microtubules (MTs). B. Regenerating protoplast showing MT organization atthe future PPB site. C. Two regenerating protoplasts each with one PPB. The individual MT strands are densely packed at thePPB site. D. A PPB that divides cell asymmetrically. Note that the nucleus is not centred on the plane defined by the PPB.E. Two daughter cells in interphase. MTs are mainly cortical. F-G. Two overlapping cells with PPBs perpendicular to eachother. When focusing at the bottom of the top cell, the two PPBs are seen as a cross (F). When focusing at the median plane ofthe top cell, only one PPB is visible (G). H. Two daughter cells with parallel PPBs.

For immunofluorescence microscopy the term MTdenotes a microtubule strand containing one or moreMTs. During early regeneration of cells from proto-plasts, the MTs became oriented. At the site of a futurePPB in dividing cells, MTs became parallel to each other(Fig. 2B) and then a PPB appeared (Fig. 2C). UsuallyPPBs divided cells symmetrically; however, some PPBswere oriented asymmetrically (Fig. 2D). In such cells,the centre of the nucleus was not necessarily in the planeof the PPB. After the first divisions MTs were mainlycortical during interphase (Fig. 2E). Some MTs deeperin the cytoplasm were out of focus but contributed to theoverall fluorescence. Newly formed daughter cells oftendivided synchronously. The orientation of the two PPBsin these cells appeared random, ranging from beingperpendicular to each other (Fig. 2F-G) to being on thesame plane (Fig. 2H).

Quantification of PPBsQuantification of PPBs in the soybean protoplast cultureswas facilitated by good staining of MTs, the presence ofindividual protoplasts and dispersed cells and high fre-quencies of PPBs. Data for four parameters are presentedin Table 1. All parameters had their highest values at 0-5day of culture and decreased at 1 -0 day. All cultures had aPPB index >1 although it varied with the time of culture.

Table 1. PPB index, % of PPB cells, mitotic index andPPB value of soybean protoplast cultures

Days afterculture

0-51-01-52-02-53-03-55-0

PPBindex

7-10± 1-731-38 ± 0 - 1 53-88 ±0-793-53 ±0-593-58 ±0-754-57 ±0-764-19 ±0-323-94 ± 1 1 8

%ofPPB cells

31-3 ± 1-17-2±0-7

20-7 ±2-212-5 ± 1-69-0 ± 1-16-5 ± 1 - 16-0 ±0-84-8 ±0-8

Mitoticindex

7-9 ±0-6S-8± 1-28-5 ±2-84-9 ±0-83-710-73-1 ±0-32-2 ±0-52-0 ±0-3

PPBvalue

4-00 ±0-411-26 ± 0 1 72-60±0-712-63 ±0-812-50 ±0-342-25 ±0-402-76 ±0-902-48 ±0-62

To obtain the PPB index, PPBs and phragmoplasts were countedto a sum of 100 in each of five or more samples and the ratios wereindividually calculated. Five hundred cells were counted for the % ofPPB cells and mitotic index in each of several samples and the PPBvalue (ratio between % of PPB cells and mitotic index) was derivedfrom them.

The PPB index was lowest for the 1-day culture. Itstabilized after 1-5 days of culture, retaining a valuebetween 3-5 and 4-5. The % of PPB cells showed asecond peak at 1-5 days and then decreased graduallybecause the % of mitotic cells decreased. PPB values weresmaller than PPB indices in general (Table 1) because thelatter were based only on the number of phragmoplastsobserved. However, the PPB value showed a trend

Soybean protoplast preprophase bands 577

Fig. 3. Development of preprophase bands (PPBs) during cell cycle of soybean protoplast cultures. The first two micrographsof each series illustrate microtubule (MT) distribution at surface (A'-F1) and median planes of cells (A"-F"). The thirdmicrographs (Am-Fm) show the Hoechst-stained nucleus of the same cell. X650. A-C. Early stages of PPB development withuncondensed chromatin. A. Early 1: a MT-rich area is evident at the site of a future PPB. B. Early 2: PPBs appearing in 2cells. C. Early 3: perinuclear fluorescence (PNF) consisting of radiating MTs is visible. The PPB is well defined. D - F . Latestages of PPB development with advanced degrees of chromatin condensation. D. Late 1: PNF is more prominent andchromatin condensation is visible. E. Late 2: the PNF is an intensely stained layer on the nuclear surface and the PPB isnarrow. The cytoplasmic fluorescence has disappeared. F. Late 3: the PNF consisting of clearly recognizable MT strands ispolarized, forming 2 foci on the nuclear surface. Only a trace of the PPB can be seen (arrowheads).

similar to the PPB index. The coefficients of variationfrom the two methods were not significantly different;however, the sample size used for estimating the PPBvalues was smaller than for the PPB indices. On average4500 cells were screened per treatment for the PPB indexwhile only 1900 cells were screened for the PPB value.

Development of PPBs during the cell cycleThe morphology of PPBs changed as division progressed.During PPB development, changes also occurred in thenucleus, cytoplasmic fluorescence and perinuclear fluor-escence (PNF). On the basis of these changes, PPBscould be classified into different stages of development.The two main stages, an early stage (preprophase) and alate stage (prophase), are described. The classificationprovided below is arbitrary, since the changes are gradualand continuous.

Early stage. Nuclei contained uncondensed chromatin

(Fig. S A ^ B ^ C " ) . Nucleoli were clearly visible and haddistinct margins.

Early 1 (Fig. 3A). A MT-rich area appeared at the siteof a future PPB. This area was relatively wide andcontained only poorly organized MTs. Some MTs wereobserved in other areas. PNF was absent.

Early 2 (Fig. 3B). The PPB was more organized andcontained mainly one layer of parallel MT strands.Faintly stained PNF appeared in some cells.

Early 3 (Fig. 3C). PPBs were well defined. PNF wasevident and consisted of MTs radiating from the nuclearsurface. In some cells, MT connections were observedbetween the PNF and PPB.

Late stage. Nuclei contained condensed chromatin andwere identified as prophase.

Late 1 (Fig. 3D). Nucleoli were still evident. The PNFwas prominent and becoming compact on the nuclearsurface.

578 H. Wang et al.

Late 2 (Fig. 3E). Cytoplasmic fluorescence diminishedso that cells appeared darker except for the PNF andPPB. PPBs became narrower and their fluorescenceappeared fainter. The PNF consisted of a thin but denselayer of MTs on the nuclear surface.

Late 3 (Fig. 3F). Chromatin was highly condensed andPPBs had almost or completely disappeared. The PNFwas often polarized and appeared as a prophase spindle.In some cells the PNF extended inside the nuclei,indicating breakdown of the nuclear envelope.

Most cells (82%) with a PPB contained interphasenuclei and the remaining 18 % had prophase nuclei. Cellsat late 3 stage contained only residual PPBs or no PPBs atall. Thus they often were not included in the cells with aPPB when counting the percentage. The PNF consistedof many recognizable MT strands rather than diffusefluorescence even at its early stage.

Discussion

PPBs were reported in protoplast cultures of Viciahajastana (Simmonds, 1986). However, these proto-plasts were capable of only very limited divisions andmost (97%) PPBs were associated with prophase nuclei.Simmonds thus referred to the bands as prophase bands.In our study of soybean protoplast cultures, more typicalPPBs formed. Up to 3 1 % of regenerating protoplastscontained a PPB after 12 h of culture. This percentage isthe highest ever reported for cultured cells and proto-plasts. Numerous PPBs were also observed during sub-sequent cell divisions. A previous study of the same cellline by electron microscopy did not reveal the existence ofPPBs in protoplast cultures (Fowke et al. 1974). Thismay be due to differences in isolation methods, loss ofmicrotubules due to the fixation techniques, limitationsof sample size with electron microscopy or changes in thecultures with time.

Gorst et al. (1986) suggested that cells from organizedtissues have a PPB index > 1 . PPBs have previously beendescribed in other cultured cells (Simmonds & Setter-field, 1983; Simmonds, 1986; Doonan et al. 1987;Hogan, 1988). However, neither quantitative data on thefrequency of PPBs nor information regarding the regen-erability of these cultures were reported. Hogan (1988)did note that in a non-embryogenic suspension culture ofthe monocot, Spartina pectina, PPBs were observed inall prophase cells stained and he suggested that cells ofthis suspension culture were potentially embryogenic,according to the proposal of Gorst et al. (1986).

The soybean cell line used in the present study hasnever undergone embryogenesis or organogenesis in the20 years since it was established. However, all protoplastcultures exhibited PPB indices > 1. Therefore, for thiscell line, high frequencies of PPBs are apparently notrelated to organized growth or morphogenesis. Athorough study of PPB frequencies in regenerable andnon-regenerable cell lines is necessary to clarify therelationship between PPB index and morphogenic poten-tial.

In depth study of PPB development using immunoflu-

orescence microscopy has only been reported in Alliumroot-tip cells (Wick et al. 1981; Wick & Duniec, 1983;Gunning & Wick, 1985). In these studies, details of PPBdevelopment were provided for two stages (early andmid) and nuclei were not concomitantly stained. Thecurrent study presents a more complete description ofPPB development and characterizes the correspondingchanges in PNF and nuclear organization. The six stagesidentified present the complete sequence of gradualchanges of PPB development, from early MT organiz-ation to the disappearance of the band. This classificationscheme should prove valuable. For instance, it willpermit rapid determination of the development stage ofthe PPB in a cell. The scheme should also facilitatecomparisons of PPBs in different cell types and species.

There are differences between the present results andthose of Wick & Duniec (1983). For example, PNFappeared later than PPBs in soybean protoplast cultures,while in onion root-tip cells they appeared simul-taneously. Also, PNF during the early development inonion root-tip cells was seen as diffuse fluorescence andthe authors suggested that this might be due to non-polymerized tubulin bound to the nuclear envelope. Incontrast, the early PNF in soybean protoplast culturesconsisted of MTs radiating from the nuclear surface andwith further development the MTs were packed gradu-ally on the nuclear envelope. These variations may reflectspecies differences, but are more likely to be due todifferences in staining procedures. The procedure usedby Wick & Duniec (1983) included squashing cells ontocoverslips, air-drying the cells and digestion of the cellwall with enzymes. In our experience, this type ofenzyme treatment reduces the clarity of microtubulestaining in the protoplast cultures. The squashing andair-drying apparently distort the original orientation ofMTs. For example, striations of PPBs as noted by Wick& Duniec (1983) are probably due to squashing of cells;similar artefacts were not observed in soybean protoplastcultures. The procedure used in the present studyavoided these harsh treatments.

In an attempt to quantify the occurrence of PPBs, anumber of parameters were determined. The PPB index(ratio between number of PPBs and number of phragmo-plasts) has been described (Gorst et al. 1986). A newparameter, the PPB value (ratio between % of PPB cellsand mitotic cells), has been determined and comparedwith the PPB index in soybean protoplast cultures. ThePPB value may be a more convenient parameter forstudying the distribution of PPBs in plants for a numberof reasons. First, the frequency of PPBs is related moredirectly to the cell cycle, a relatively constant character-istic of a particular cell type. This should eliminatevariation due to time taken to complete any one stage ofmitosis (e.g. cytokinesis). Estimates by both methodsshowed similar trends and variation, despite the muchsmaller sample size used for the PPB value. Thus datacollection for the PPB value is generally less time-consuming. Finally, one set of data collected for the PPBvalue provides direct information regarding the absolutefrequency of PPBs in a cell population (i.e.% PPB cells).Such information is not available from the PPB index.

Soybean protoplast preprophase bands 579

The soybean protoplasts exhibited partially synchron-ous cell division, similar to that reported by Weber et al.(1986). All the parameters measured had highest valuesat 0-5 day of culture and decreased dramatically at 1-0day. The availability of PPBs in high frequencies shouldbe useful for further study of this unique plant structure.

The authors are grateful to Ms M. Loewen for excellenttechnical assistance and Mr D. Dyck for photographic assist-ance. An operating grant (A6304) from the Natural Sciencesand Engineering Research Council of Canada to L.C.F. isgratefully acknowledged. H.W. is supported by a scholarshipfrom the University of Saskatchewan.

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(Received 20 October 1988 - Accepted 13 January 1989)

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