Cell biology of the stigma of Brassica campestris in relation to C02

effects on self-pollination

PATRICIA O'NEILL, MOHAN B. SINGH and R. B. KNOX

l'lnnt Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia

Summary

The effect of carbon dioxide in blocking thesporophytic self-incompatibility system in Bras-sica campestris occurs within the first 2h ofpollination at the pollen-stigma interface. Per-centage germination of self pollen on the stigmawas found to be similar in air and in the presenceof 5 % CO2. The CO2 effect therefore must occurafter pollen germination, modifying the interac-tion between pollen tubes and stigma cells. Lectinbinding studies showed the presence of fucosylbut not galactosyl residues on the stigma surface.

Gel electrophoresis of plant extracts showed thatstigma esterase activity is marked in comparisonto other plant tissue. This activity is shown histo-chemically to be localized on the stigma cellsurface and in the nucleus. Carbonic anhydrasehas been detected on the stigma surface by twodifferent histochemical methods and its possiblerelationship to the CO2 effect is discussed.

Key words: self-incompatibility, esterase, lectins, carbonicanhydrase.

Introduction

Brassica species have been shown to have a sporo-phytic self-incompatibility system in which pollen ofthe same S (self-incompatibility) genotype as thefemale parent is rejected at the stigma surface (seereview by Heslop-Harrison, 1978). Carbon dioxideadded to the environment during a self-pollination hasbeen shown to induce seed set in self-incompatibleBrassica genotypes (Nakanishi et al. 1969). The for-mation of the polysaccharide callose in the stigmapapillae, characteristic of self-pollen rejection, isreduced in the presence of 5 % CO2 (O'Neill et al.1984).

The effect of CO2 in overcoming self-incompatibilityhas been monitored cytologically. After 24 h of 5%CO2 treatment, pollen tubes could be seen in squashpreparations of self-pollinated styles of B. campestris(O'Neill et. al. 1984). Stigma penetration by pollentubes has been shown to occur in the first 2h afterpollination and treating self-pollinated stigmas with5 % CO2 later than 3 h after pollination causes noincrease in pollen tube penetration (Nakanishi &Hinata, 1973). Therefore it may be assumed that CO2

Journal of Cell Science 89, 541-549 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

is acting at the stigma surface during the first 2 h afterpollination, on either the pollen grains or the pollentube interaction with the stigma papillae.

CO2 may be changing the activity of a stigmaenzyme. Esterase activity towards the synthetic sub-strate a"-naphthyl acetate has been known for manyyears to be present at the stigma surface, and has alsobeen shown to be an indicator of stigma receptivity(Mattsson et al. 1974). In animal systems esteraseactivity towards the substrate jS-naphthyl acetate hasbeen shown to indicate the presence of carbonicanhydrase (Tashian, 1969). The observation that CO2affects incompatibility at the stigma surface and thisobservation of esterase activity led to the hypothesisthat CO2 may be affecting carbonic anhydrase activity.

In this report, the location of the CO2 effect isdeduced from experimental pollinations carried out inthe presence and absence of 5 % CO2. On the basis ofthis observation, various surface polysaccharide com-ponents were probed. Esterase activity in the stigmacells was characterized and observed under variouspollination treatments and its relationship with car-bonic anhydrase was explored.

541

Materials and methods

Plant materialPlants of Brassica campestris var. T.1S of self-incompati-bility genotypes S)S| and S2S2 (our own nomenclature) wereraised to flowering in a glasshouse and then transferred into agrowth cabinet with a 14/10h, day/night, and 20°C/l5°Ctemperature regime. After a week of acclimatization, flowerswere emasculated at yellow bud stage and allowed to open.For pollination and CO2 treatment, open flowers weredetached from the plants and mounted in 1 % agar. Immedi-ately after pollination the pistils mounted in agar were placedin plastic chambers through which air or S % CO2 in air(Commonwealth Industrial Gases) was passed. The air andCC^/air mix were both bubbled through a saturated solutionof NaCl to keep humidity at 76% r.h. as humidity levelsabove 90 % r.h. have been shown to affect the self-incompati-bility response in Brassica (Carter & McNeilly, 1975). Pistilswere left in these conditions for 2h and were then removedfrom their respective chambers and left for a further 2 h in thecabinet prior to processing. S2S2 stigmas were used in allpollinations with either S2S2 pollen (self) or S1S1 pollen(cross).

For gel electrophoresis of plant parts, the wild-typecollection of B. campestris var. T.1S was used.

Pollen germinationTen flowers from four treatments: (1) self-pollinated,(2) self-pollinated + CO2, (3) cross-pollinated and (4) cross-pollinated + CO2, were fixed after the 4-h treatment de-scribed above and processed for pollen tube observation(Kho & Baer, 1968). The total number of pollen grains andthe number of germinating pollen grains on each stigma werecounted and expressed as a fraction of pollen germination(germinating grains/total no. of grains) on the stigma. Themeans of each set of 10 values and standard errors of eachmean were calculated. Pollen viability was found to be greaterthan 95 % as measured by the fluorochromatic reaction(Ileslop-Harrison & Heslop-Harrison, 1970).

Pollination cytochemistryB. campestris pistils from four different treatments:(1) unpollinated, (2) self-pollinated, (3) cross-pollinated and(4) self-pollinated + CO2, were lightly fixed in 2% freshlydepolymerized paraformaldehyde, 0-5% glutaraldehyde in005M-Pipes-NaOH buffer, pH8, for 2h at room tempera-ture and rinsed in 015M-Pipes-NaOH buffer, p H 6 8 , at4°C overnight. The samples were then dehydrated throughan ethanol series and transferred into London Resin White(London Resin Company), infiltrated over 2 days andembedded at 60°C for6h.

Blocks were sectioned at 2ftm and sections mounted onmicroscope slides in drops of dilute Mayer's egg albumin.General observations of stigma papilla cytochemistry weremade using Mallory's Azur II—Methylene Blue stain(Richardson el al. 1960).

Relative DNA estimationUnpollinated stigma semithin sections (2/um in thickness)were stained with the DNA fluorochrome DAPI (4,6-

diamidino-2-phenylindole) according to the method of Cole-man & Goff (1985), and were used to calculate the DNAlevels in the nuclei of stigma papillae compared with theunderlying stigmatic zone cells. The amount of fluorescenceemitted by the nuclei of each type was measured using a CarlZeiss Microscope Fluorometer, using an aperture diameter of15ftm around each sectioned nucleus. The fluorescentemissions contributed by each nucleus under the ultraviolet(u.v.) filter combination were estimated in arbitrary units,with the background set at zero. One hundred fluorescencemeasurements of cells were made for each nuclear type, fromstigmas within the same semithin section to ensure uniform-ity of thickness and staining.

The area of each nucleus and putative volume, assuming anellipsoidal shape, was determined by recording the micro-scopic image by means of a video camera linked to the videoscreen of the Carl Zeiss Videoplan Image-analysis Systemusing the TV-overlay board. Fifty nuclei of each type weredigitized to compute nuclear area of the semithin sections.

The mean area for each type of stigma nucleus wasmultiplied by the thickness of the section (2,um) to calculatethe volume of the section contributing the DAPI fluor-escence. The amount of fluorescence in the total volume ofeach nucleus was then calculated for each group. The relativeDAPI fluorescence of the stigma papillar nuclei to that of thestigmatic zone nuclei was calculated as a relative measure ofDNA content between stigma papillae, as follows:

Fluorescencelola| =fluorescencescct;on X volume,ola|

volumeseclion

Lectin bindingLectins tested were Ulex europaeiis agglutinin type 1 (UEA),Riciims communis agglutinin type 1 (RCA), soybean ag-glutinin (SBA) and peanut agglutinin (PGA), all linked withfluorescein isothiocyanate (FITC) and made up to 1 mgrnl"'in 001 M-phosphate-buffered saline (0-015 M-NaCl). FITC-labelled concanavalin A (ConA) made up to lmgml" 1 in0-005 M-Tris-buffered saline (0-015 M-NaCl, 001 M-CaCl2,0-01 M-M11CI2) was also tested (see Clarke & Hoggart, 1982).All lectins were supplied by E.Y. Laboratories, San Mateo,CA, USA.

Emasculated and detached S2S2 flowers were checkedthoroughly for pollen contamination. Pistils were thenupturned and their stigmas immersed in various FITC-labelled lectin solutions in a microtitre tray for 30min.Stigmas were then rinsed in the appropriate buffers for 5 minand observed under u.v. fluorescence microscopy. Wherelectin binding was observed, control stigmas were treatedwith lectins in the presence of the appropriate hapten sugars.

Gel electrophoresisStigma, style/ovary, pollen and leaf tissues from the wild-type plants of B. campestris were collected and stored at—20°C. A weighed amount of frozen plant material wasextracted on ice in 0 1 M-Tris-borate containing 001 M-dithiothreitol, 001 M-disodium ethylenediaminetetra-acetateand 0001 M-phenylmethylsulphonyl fluoride. The proteincontent of each extract was assayed according to Bradford(1976) with bovine serum albumin as a standard.

542 P. O'Neill et al.

A 50 fig sample of protein from B. campestris stigma,ovary/style, pollen and leaf extracts was loaded onto a 10 % toIS % SDS-polyacrylamide gel, run at 20 mA for 1 h and thenat 30 mA for 3h at 4°C. The gel was then washed over 1 hwith four rinses of 20% isopropanol in 001 M-Tris- H Obuffer to renature enzymes (Blank et al. 1983) and thenstained for esterase activity towards the substrates a- and /3-naphthyl acetate as follows: 2ml of a 1 % solution of u/f5-naphthyl acetate (Sigma) in 50 % acetone was added to 25 ml0-4M-THS-I-IC1 buffer, pH7-0, and made up to 100ml withdistilled water. A lOOmg sample of Fast Blue RR salt wasadded and the mixture was filtered before immediate use(Tashian, 1969). This solution stained af-naphthyl acetateesterase bands black or /J-naphthyl acetate sites red.

A 100 fig sample of protein from extracts of B. campestrispollen and stigma was run, on a similar gel to that describedpreviously, against low molecular weight markers. From thisgel the approximate molecular weights of the esterase-stainedbands were assessed by overstating with Coomassie Blueprotein stain.

Enzyme histochemistry

Esterase (EC 3.1.1.2) was localized on frozen and plasticsemithin sections of B. campestris unpollinated and polli-nated stigmas. These were incubated, in the same esterasereaction mixture, described above, for 15min. They werethen rinsed thoroughly, mounted in 50 % glycerol andobserved microscopically. Controls were carried out usingthe reaction mixture without the addition of the syntheticsubstrate.

Two methods for the localization of carbonic anhydrase(EC 4.2.1.1) were used. (1) The technique of Pochhammeret al. (1979): whole fresh unpollinated pistils of B. campestriswere incubated in the following solution for 1 h. 5-Dimethyl-amino-1-naphthalene sulphonamide (dansylamide, Sigma)was dissolved in acetone and added to 0-067 M-phosphatebuffer, pH7-4, to give 3x l0~ s M-dansylamide. Pistils werethen rinsed thoroughly, mounted in buffer and whole stigmaswere observed under u.v. fluorescence microscopy. Controlstigmas were preincubated in 10~ M-acetazolamide (Sigma)prior to incubation in dansylamide solution also containing10~5 M-acetazolamide. (2) The technique of Hansson (1967):whole fresh unpollinated pistils of B. campestris were slitlongitudinally and placed in the following reaction mixture:6-0 ml of a 0-05 M-H2SO4 solution was added to 0-1 M-CoSO4;l-0g NaHCO3 was dissolved in 50 ml 0-lM-Na2SO4 andimmediately added to the COSO4 solution. Pistils wereincubated in this solution in Petri dishes for 1 h, rinsed in10% sucrose, stained in 1 % (NH4)2S for 1 min, rinsed andmounted in 10% sucrose for microscopy. Controls includedpreincubation of pistils in 10~ M-acetazolamide and theomission of NaHCOj from the reaction medium.

Results

The carbon dioxide effect

The results of measurements of pollen germinationafter experimental CO2 treatment for 2h are given inTable 1. It can be seen that when CO2 is added to theenvironment during self- or cross-pollinations, pollen

Table 1. Effect of CO2 treatment on pollengennination

Self-pollinatedS2S2 X S2S2

Cross-pollinatedO2S2 X S) S |

Air5 % CO2 in air

0-225 ±0-140-183 ±0-10

0-83510-120-782 ±0-10

Each value is a mean of 10 values calculated as no. ofgerminating pollen grains/total no. of pollen grains for each of 10stigmas per treatment ± standard error.

germination does not change significantly. About 80 %of pollen was seen to germinate on cross-pollinatedstigmas but only 20 % germination was observed onself-pollinated stigmas, irrespective of CO2 treatment.Therefore, since CO2 treatment does not result in anincrease in the amount of germination on self-polli-nated stigmas, it follows that CO2 must be acting on thepoint at which pollen tubes interact with the stigmapapillae and allowing penetration of pollen tubes whereit would normally be inhibited.

Pollination histochemistry

Semithin sections of unpollinated, self-pollinated,cross-pollinated stigmas, and self-pollinated stigmas inthe presence of CO2, can be seen in Fig. 1. Theappearance of unpollinated (Fig. 1A) and self-polli-nated (Fig. 1C) stigma papillae is similar, each contain-ing a large convoluted vacuole and a large multinu-cleolar nucleus. These nuclei fluoresce brightly whenstained with the DNA fluorochrome DAPI (Fig. IB)and the papillar nuclei occupy a greater surface areathan the nuclei at the subtending stigma cells(Table 2). The nuclear volume, assuming an ellip-soidal shape, is very different for each cell type, thestigma papillar nuclei being 12 times greater than thoseof the cells below in the stigmatic zone. Microfluor-imetry showed that the relative DNA content (esti-mated from DAPI fluorescence) of the papillar nuclei isnearly six times that of the stigmatic zone nuclei(Table 2).

Table 2. Relative DNA content of stigmatic cells ofB. campestris determined by microfluorimetiy of

semithin sections stained with the DNA fluorochromeDAPI

Stigmatic papillae Stigmatic zone

Fluoresccncesection

Nuclear area (/<m2)Nuclear volume (/«m3)Fluorescencetota|Relative DNA content

Values are expressed

8888

571285

•612-0•613-71 136-4

•6

5-9:1-0

as means i standard errors.

34164548

•41 1• 2 1 0• 4 1 2•3

•2•5•1

Stigmas of Brassica 543

Fig. 1. Light micrographs oflongitudinal sections throughstigmatic papillae of B. campestris.sp, stigma papilla; n, nucleus;pg, pollen grain; pi, pollen tube.A. Unpollinated, Richardson'sstain. X700; B, unpollinated,DAPI stain. X350; C, self-pollinated, Richardson's stain.X700; D, self-pollinated + CO2

treatment, Richardson's stain.X700.

Self-pollinated stigmas in the presence of CO2(Fig. ID) have been penetrated by pollen tubes. Thisprocess results in a breakdown of the papillar wall andplasma membrane and disruption of cytoplasmic con-tents. The breakdown of the large stigma nuclei isapparent following pollen tube penetration into thepapillae. Pollen grains on the surface in both cross- (notillustrated) and self- + CO2 pollinations have collapsedconsiderably due to the utilization of their contents inthe process of germination and penetration. Overall,the self- + CO2 interaction is cytologically similar to anormal compatible pollination.

Lectin binding

FITC-labelled RCA, SBA and PNA lectins, specificfor galactose-related residues did not bind to unpolli-nated B. campestris stigmas. FITC-labelled ConA(specific for cv-L-mannosyl and a'-D-glucosyl residues)bound to the papillae (see Fig. 2A) but was notcompletely inhibited when the labelled ConA waspreincubated with the specific inhibitor, 0-2M-ff-D-methylmannopyranoside (Fig. 2B).

FITC-labelled UEA lectin bound strongly to thepapillae (Fig. 2C) and binding was much reducedwhen UEA was preincubated with 0-2M-a/-L-fucose

544 P. O'Neill et al.

Fig. 2. Fluorescent-labelledlectin binding to freshB. campestris stigmas. X640.A, FITC-labelled ConA;B, control in which F I T C -ConA was pre-incubatedwith 0-2M-a'-D-methylmannopyranoside;C, FITC-labelled UEA;D, control in whichFITC-UEA was pre-incubated with 0-2M-CV-L-fucose.

(Fig. 2D). Of the sugar residues tested, fucose appearsto be a major sugar component of the stigma surface.

Stigma esterase

Gel electrophoresis of extracts from various tissues ofB. campestris showed esterase activity towards thesubstrate cv-naphthyl acetate in two highly active blackbands in stigma tissue (Fig. 3, lane a). The upper bandwas also detected in ovary/style and pollen (Fig. 3,lanes c,d) whereas the lower band appeared to bestigma-specific. These two active stigma bands haveapproximate molecular weights of 31X103 and 34X103

(Fig. 3, lanes e,f). One red band of esterase activity at34xlO3Mr towards the substrate jS-naphthyl acetatehas been detected in the stigma extract (Fig. 3, lane g).

Semithin plastic sections of B. campestris pistilsshowed a highly specific esterase reaction towards thesubstrate cv-naphthyl acetate (see Fig. 4). Only thestigma papillae area of the pistil section stained with the

reaction mixture. Unpollinated stigmas showed a darkreaction in stigma walls and nuclei, particularly in thenucleoli (Fig. 4A). When unpollinated sections wereincubated in the reaction mixture without the substratecv-naphthyl acetate, no coloration of the stigma cellswas observed (Fig. 4B). /3-Naphthyl acetate esteraseactivity was also found in semithin sections of B.campestris stigma, but the red reaction product wasmore diffuse than the black reaction product observedfor cv-naphthyl acetate.

Stigma carbonic anhydrase

Stigmas incubated in the fluorescent substrate dansyl-amide showed a concentrated accumulation of thisfluorescent complex on the outer surface of the papillae(see Fig. 5A). Preincubation of stigmas in 10~5M-acetazolamide produced diffuse fluorescence, indi-cating that binding was reduced although completeinhibition did not occur (Fig. 5B).

Stigmas o/Brassica 545

HX1(T3

- 9 4

i

-•67

- 4 3

- 3 0

- 2 0 - 1

— 14 1a b e d e f 9 h

Fig. 3. Characterization of esteraseactivity in stigma extracts ofB. campestris using SDS-gelelectrophoresis. Lanes a-d stained foresterase activity towards the substratea--naphthyl acetate. Lanes e-h stainedwith Coomassie Blue. Lane a. Stigmaextract showing two esterase bands(arrows) at 31X10"3 and 34xlO"3/l/ r;b, leaf extract; c, ovary/style extract;d, pollen extract; e, molecular weightmarkers (XlCT3): a--lactalbumin (14-4),soybean trypsin inhibitor (20'1),carbonic anhydrase (30), ovalbumin(43), bovine serum albumin (67) andphosphorylase (94); f, same as a butoverstained for protein; g, esteraseactivity towards the substrate)3-naphthyl acetate. Positive red band isarrowed; h, stigma extract stained forproteins.

4A

Fig. 4. Esterase activity withcv-naphthyl acetate as substratein longitudinal sectionsthrough stigmatic papillae ofB. campestris. X700.A. Unpollinated;B, unpollinated controlwithout (r-naphthyl acetate.

546 P. O'Neill el al.

Fig. 5. Carbonic anhydrase activity in fresh B. campestris stigmas. A. Dansylamide treated. X640; B, control for A in thepresence of 10~5M-acetazolamide. X640; C, Hansson's reaction. x320; D, Hansson's reaction. X640; E, control for Dwithout the addition of NallCOj to the reaction medium. X640.

The black cobalt sulphide reaction product indi-cating the presence of carbonic anhydrase by Hansson'smethod appeared to be restricted to the stigma papillarsurface (see Fig. 5C,D). The tissue exposed by slittingthe style longitudinally did not show binding of thereaction product. Control pistils incubated without theaddition of NaHCC>3 showed no reaction (Fig. 5E).Again, the presence of 10~5M-acetazolamide did notcompletely inhibit the enzyme reaction (not illus-trated).

Discussion

The sporophytic self-incompatibility system shown byB. campestris requires that self pollen grains berejected by the plant at the stigma surface. This surfacemust therefore possess certain macromolecules thatdetermine whether a pollen grain is accepted orrejected. Specialized large papillate cells have evolvedin Brassica to house this reaction and they have beenshown to secrete a protein surface layer termed apellicle (Mattsson et al. 1974). Synthesis of this uniquesurface pellicle requires highly specialized mechanismsfor protein production and secretion.

One way to carry out this specialized synthesis wouldbe to increase the amount of DNA contained in thesestigma papillae. Microfluorimetric estimates of DNAcontent showed that the stigma papillae have six timesmore DNA than that of the underlying stigmatic zonecells. The sizes of the nuclei and nucleoli of the papillaewere also shown to be greater to allow for the extrabiosynthetic demands on these cells. It is difficult todetermine whether this increased DNA content rep-resents amplification of selected regions of the genome.However, it is possible that the nucleolar size increasemay reflect selective amplification of ribosomal DNA,which has been observed in other secretory cells (Nagl,1974). Thus, the nucleo-cytoplasmic machinery of thepapillae appears to equip the stigma well for its specialrole in pollination.

One class of macromolecules found in stigma pa-pillae is the S-specific glycoproteins, which have beenshown to be produced exclusively by the stigma andvary between plants of different S genotype (Nishio &Hinata, 1982). These molecules are thought to act inthe self-pollen rejection response although the mechan-ism of action has not yet been confirmed. Severalstigma surface components have been localized in thisstudy.

Stigmas of Brassica 547

Probing the stigma for surface polysaccharides orglycoprotein sugar residues revealed binding of thelectin Con A. This binding was found to be non-specificas it was not completely inhibited by the specific haptensugar. Gaude & Dumas (1986), in their electronmicroscope study of B. oleracea stigma surface com-ponents, also found non-specific binding of Con A, withthe same electron density of binding observed intreatments and controls. UEA lectin binding to thestigma surface, however, was inhibited specifically byits hapten sugar, fucose, indicating the presence offucosyl residues in the stigma pellicle. Nasrallah(1987), in an analysis of Brassica stigma glycoproteins,found a significant fucosyl component. Our datasuggest that these fucose-containing glycoproteins maybe present in the stigma pellicle.

The location of esterase activity in the stigma sur-face, confirmed in this study, suggests a possible rolefor esterase in compatible pollination. Knox et al.(1975) suggested that the pollen esterase may complexwith stigma esterase to form an active 'cutinase' todigest the stigma cuticle and enable pollen entry intothe stigma apoplast. Ferrari etal. (1985) suggested thatesterases on the papillar surface are ideally sited andfunctionally capable of hydrolysing pollen coat oils,perhaps to cross-link pollen and stigma surfaces toassist in pollen grain adhesion.

Comparative gel electrophoresis revealed a stigma-specific esterase isozyme that was not detected in leaf,ovary or pollen protein extracts. It was not possible toconclude whether this unique form of esterase isrestricted to the papillae or is found in all the cells ofthe stigma.

Another component of the stigma surface enzymeactivity is carbonic anhydrase, as shown by the histo-chemical reactions in fresh unpollinated stigmas. Lackof complete inhibition with acetazolamide means thatthe specificity of this binding needs further investi-gation. It has been reported that sulphonamides, suchas acetazolamide, are about 1000 times less inhibitoryto plant carbonic anhydrases than to erythrocyte car-bonic anhydrase (Atkins et al. 1972), so an alternativecontrol inhibitor for stigma carbonic anhydrase isrequired.

The presence of this stigma carbonic anhydrase maybe important when considering the effect of CO2 inremoving the self-incompatibility rejection response.Raised levels of CO2 (3-5 %, v/v, in air) have beenshown to suppress the activity of carbonic anhydrase inthe green alga Chlamydomonas (Kimpel et al. 1983)and the blue-green alga Anabaena (Shiriawa & Mi-yachi, 1985). Since carbonic anhydrase is one of thefew enzymes directly affected by CO2 concentration, itcould be involved in the breakdown of sporophytic self-incompatibility by CO2.

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{Received 14 July 19S7 -Accepted, in revisedfonn,14 December 19S7)

Stigmas of Brassica 549