CHARACTERISTICS OF POLLEN DIFFUSATES AND POLLEN WALL CYTOCHEMISTRY IN POPLARS

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1 Cell Sri. 44, 1-17 (1980) Printed in Great Britain Company of Biologists Limited ig8o CHARACTERISTICS OF POLLEN DIFFUSATES AND POLLEN WALL CYTOCHEMISTRY IN POPLARS A. E. ASHFORD AND R. B. KNOX School of Botany, University of Neio South Wales, Kensington, N.S.W. 2033, and School of Botany, University of Melbourne, Parkville, Victoria, 3052, Australia SUMMARY Pollen diffusate8, known to be important in pollen-stigma interactions controlling interspecific incompatibility between Populus deltoides and Populus alba, have been partly characterized and shown to contain more than 20 protein bands by polyacrylamide gel electrophoresis, at least 4 of these being glycoproteins. Seven fractions had antigenic activity in rabbits and several enzyme activities were also present. Peroxidase and leucine aminopeptidase isoenzymes were detected in the diffusates, demonstrating the extracellular location of these 2 enzymes. Isoenzyme patterns of peroxidase, esterase and acid phosphatase were complex, with some bands common to both species. Localization of acid phosphatase in the intine and esterase in the exine was demonstrated after brief aldehyde fixation and low-temperature embedding in glycol methacrylate. The intine and exine sites were distinguished by their chemical and structural features. Calcofluor white M2R new proved to be an excellent stain for differentiating the intine. Aniline blue-positive material, probably /?-i,3-glucan, is present associated with the intine of many ungerminated as well as germinating grains: production of this material may be a response to damage. INTRODUCTION Pollination experiments between Populus deltoides, the American black cottonwood and P. alba, the white poplar, provided the first evidence that extracellular pollen-wall proteins act in the recognition reactions that determine interspecific incompatibility between these 2 species (Knox, Willing & Pryor, 1972; Knox, Willing & Ashford, 1972). When killed self-compatible pollen or self pollen-wall protein diffusates were applied to P. deltoides stigmas, followed by incompatible P. alba pollen, hybrid seeds were produced. In the absence of the killed self pollen or diffusate, no seed set occurred. These observations have since been extended to sporophytic self-incompatibility systems (see review by Clarke & Knox, 1978). In this paper we characterize the ' active' P. deltoides diffusates, using a variety of immunological and electrophoretic techniques and provide evidence that they originate from extracellular sites in the pollen grain. In the course of the work it was necessary to undertake a cytochemical study to define the nature of the pollen grain wall in Populus.

2 2 A. E. Ashford and R. B. Knox MATERIALS AND METHODS Pollen sources Catkin-bearing branches from trees of P. deltoides or P. alba var. bolleana, grown in the campus of the Australian National University, Canberra, were brought into the glass-house just prior to anthesis and placed in water-filled containers. Pollen release occurred over several days, and pollen which had fallen from the catkins was collected daily and either used immediately, or desiccated over silica gel for h, and stored dry at 18 C. Pollen viability was checked using the fluorochromatic test of Heslop-Harrison & Heslop-Harrison (1970). Pollen germination experiments Pollen was dusted onto ripe stigmas of either P. deltoides or P. alba, maintained in the glasshouse as 'bottle grafts' as described by Knox et al. (1972). Cytochemical methods For most cytochemical studies, plant material was fixed either in 10 % unbuffered acrolein or 3 5% glutaraldehyde buffered with M phosphate buffer at o C C, and dehydrated and embedded in glycol methacrylate (GMA) at 25 C according to the method of Feder & O'Brien (1968). For preservation of enzyme activity, tissues were embedded in GMA at low temperature ( 35 C) by the method of Ashford, Allaway & McCully (1972). Both mature ungerminated pollen and pollen germinating on stigmas were fixed and embedded for cytochemical study. Freeze-sectioning of anthers was carried out as described by Knox (1970) using gelatinglycerol embedding at 15 C. Staining procedures Toluidine blue at ph 4-4. Sections were stained for 5 min as described by Feder & O'Brien (1968). The dye solution was 005 % toluidine blue buffered with sodium acetate buffer. Periodic acid-schiff reaction. The PAS reaction was carried out as described by Feder & O'Brien (1968), using 2,4-dinitrophenylhydrazine in 15 % acetic acid for the aldehyde blockade. For controls, periodate oxidation was omitted. Sudan black B. Sections were stained for 15 min in a saturated solution of sudan black B in 70% ethanol, briefly rinsed in 70% ethanol, and mounted in glycerol-gelatin (Pearse, 1968). Auramine O. The method used is based on that described in Heslop-Harrison (1977). GMA-embedded sections were stained in 005 % auramine O in 005 M potassium phosphate buffer at ph 65 for 5 min, rinsed in distilled water, mounted in immersion oil and observed immediately. Naphthol blue black. Sections were stained in 1 % naphthol blue black in 7 % acetic acid for 10 min at room temperature and rinsed in 7 % acetic acid (Fisher, 1968). i-anilino-s-naphthalene sulphonate (ANS) stain for proteins. Fresh material was stained with o-ooi % ANS in 001 M phosphate buffer at ph 6'8, containing 15 % methanol, and then rinsed in 15% methanol (Heslop-Harrison, Knox & Heslop-Harrison, 1974). Following staining, material was observed by fluorescence microscopy. Calcofluor white M2R new for fi-linked polysaccharides. Pollen grain walls were stained with a o-i % aqueous solution of Calcofluor white M2R new (Maeda & Ishida, 1967; Hughes & McCully, 1975) and examined by fluorescence microscopy. Aniline blue reaction for fi-i,2-glucans. Sections were stained with 001 % aqueous aniline blue in phosphate buffer, ph 95, as described in Smith & McCully (1978). Following staining, GMA-embedded sections were not rinsed, but were blotted, then air dried and mounted in immersion oil for observation. Unfixed pollen grains from pollen stock stored at 18 C were examined directly in the aniline blue solution. Control sections mounted in alkaline buffer, without aniline blue, were also examined to determine autofluorescence of tissues. Esterase localization, a-naphthyl acetate was used in Burstone's simultaneous-coupling reaction either with tetrazotized O-dianisidine at ph 8-o or hexazotized pararosanilin at ph 6-5 (Pearse, 1972).

3 Characteristics of poplar pollen walls 3 Acid phosphatase localization. GMA-embedded sections were reacted with naphthol AS-BI phosphate or a-naphthyl acid phosphate and hexazotized pararosanilin at ph 6-o, according to the method of Barka & Anderson (1962). Immunofluorescence. Methods were as described in Knox (1971). Enzymic extractions. GMA-embedded sections of mature pollen were extracted with the following enzyme solutions: pectinase (Sigma No. P-4625), at 1 mg/ml, ph 40 and 25 C; hemicellulase (Sigma No. H-2125) at 10 mg/ml, ph.5-5 and 58 C; papain (Sigma No. P-3375) at 1 mg/ml, ph 6-2 and 25 C. Sections were flooded with enzyme solution, left for varying periods of time up to 48 h, then washed and stained with toluidine blue. As controls, sections were incubated in solutions without enzyme. Scanning electron microscopy Pollen samples were rinsed in ethanol:ether (1:1, v/v), dried, coated with platinum and observed in a JEOL UM3 scanning electron microscope at 15 kv. Treatment of pollen for extraction of wall proteins About 50 g of fresh pollen were suspended in 250 ml of solution containing 10 % mannitol and o-oi % calcium chloride, buffered with M Tris buffer ph 7-9. This was done at o C C. The mixture was gently shaken for 40 min after which the pollen was separated from the solution by filtration through several layers of cheesecloth. Examination of some of the pollen grains thus treated, either unfixed or after fixation and embedding in GMA by Feder & O'Brien's (1968) method, showed that the pollen grains had not burst and were not apparently structurally damaged by this treatment (Fig. 2 A, B, p. 5). Similarly, results testing the permeability of the pollen-grain membrane with ftuorescein diacetate (Heslop-Harrison & Heslop-Harrison, 1970) indicated that membrane integrity was good. In fact, this method was used to test the effect of a range of concentrations of mannitol and calcium chloride on pollen viability, resulting in the choice of the 10 % mannitol solution, which caused no detectable loss of viability. Thus, we have a ' diffusate' of pollen proteins rather than an extract. Proteins were extracted from the nitrate by ammonium sulphate precipitation, ammonium sulphate being added slowly until total saturation was achieved. The flocculent precipitate was collected by centrifugation at g for 15 min. The pellet was resuspended in a small volume (20 ml) of M Tris buffer and was desalted in 5-ml batches through a G25 Sephadex column, which also removed low-molecular-weight phenolic compounds. The protein content in each fraction collected was estimated by absorbance at 280 ran in a u.v. spectrophotometer. The protein came off as a single peak, collected in a series of fractions, which were subsequently recombined, to give a solution containing approx. 2 mg/ml protein. This protein solution was used directly, without further treatment, in electrophoretic and immunofluorescence studies. Polyacrylamide gel electrophoresis Disk electrophoresis. Polyacrylamide gel disk electrophoresis of pollen diffusates was run under similar conditions to those described in Davis (1964) using a large-pore spacer gel (2-5 % acrylamide) and a small-pore running gel (7-5 % acrylamide). The upper and lower reservoirs were filled with o-i M Tris-glycine buffer, ph 8-3. A sample gel was not used, but a suitable volume of pollen diffusate (50 fig of protein) was applied to the top of the spacer gel. Bromophenol blue was added to the buffer in the top tank to indicate the position of the moving front. Electrophoretic separation of proteins was then carried out at 4 C using a constant current of 2-5 ma/gel column. The run was terminated when the marker dye had migrated to within 2 mm of the base of the tubes. Eltctrophoresis on continuous gradient slab gels. Electrophoresis was carried out on a Gradipore Electrophoresis unit using continuous gradient (approx. 4 % to 26 % acrylamide) slab gels (supplier Townson & Mercer). The upper and lower tank reservoirs contained Tris-EDTAborate buffer (Tris, g> Naj EDTA, 0-93 g; boric acid, 5-04 g; water, 1000 ml). Samples of 15 ftl containing approx. 50 fig protein were carefully layered in slots at the top of the gel.

4 4 A. E. Ashford and R. B. Knox Electrophoretic separation of proteins was carried out at 4 C at a constant voltage of 75 V for 24 h. The current fell from 30 ma to approx. 10 ma during the first 3 h. Bromophenol blue was used to indicate the position of the moving front. Staining of the gels Total protein. Gels were stained in ro% amido black 10B in 20% acetic acid. Excess dye was removed by destaining in 7 % acetic acid for a minimum of 48 h. Polysaccharide. Gels were treated in 7 % acetic acid for 1 h prior to staining, to leach out Tris buffer. They were then stained in the following sequence: 1 % periodic acid in 3 % acetic acid, 1 h; water rinse, 1 h; Schiff's reagent, 1 h; 1 % aqueous sodium metabisulphite rinse, 1 h. Following rinsing, gels were stored in 1 % aqueous sodium metabisulphite. Leucine aminopeptidase. The simultaneous-coupling method of Nachlas, Crawford & Seligman (1957) employing L-leucyl-yS-naphthylamide hydrochloride as substrate and tetrazotized O-dianisidine (diazo blue B salt) as coupling agent was used. Concentrations of reagents were the same as in the above paper, but the ph was adjusted to 7-1. Acid phosphatase. The incubating medium contained a-naphthyl acid phosphate (1 mg/ml) and tetrazotized O-dianisidine (1 mg/ml) in 0-05 M acetate buffer at ph 5-0 (Pearse, 1968). Gels were incubated for 1-2 h. Esterase. A simultaneous coupling method was used, employing a-naphthyl acetate (o-6 mg/ml) as substrate and tetrazotized O-dianisidine (1 mg/ml) in 0025 M phosphate buffer at ph 8-0 (Pearse, 1972). Good staining was obtained in 30 min to 1 h. Peroxidase. Gels were stained by a procedure modified from that of Novikoff & Goldfischer (1969) employing diaminobenzidine tetra-hydrochloride (2 mg/ml) and hydrogen peroxide (0-02%) buffered with Tris at ph 9-2. Addition of 002 M 3-amino-i,2,4-triazole to the incubating medium did not influence the isoenzyme pattern. OBSERVATIONS Structure and cytochemistry of Populus pollen grains Populus pollen grains are non-aperturate (Erdtman, 1966) and are c. 0-30//.m in diameter (Fig. 1 A). The exine is semitectate with conspicuous crevices, and its surface has a reticulate pattern that is perforated by conspicuous micropores (Figs, IB, 5 A), while the tectum is surmounted by small spines. The intine is uniformly thickened (1-1-5 / ltn wide) all round the grain and is the dominant wall layer (Figs. 2B, 4A, 4E). In GMA-embedded sections the exine appeared as a thin irregular monolayer, which in some areas did not cover the intine (Figs. 2B, 4A, 5 A). An interpretation of wall structure in Populus, based on our observations and electron micrographs of the wall in related species (Rowley & Erdtman, 1967), is presented in Fig. 3. Various cytochemical tests were used to differentiate the exine and intine layers. Following toluidine blue staining (ph 4-4) in GMA-embedded P. deltoides pollen sections, the exine stained bright green, indicating that phenolic materials may be present (O'Brien, Feder & McCully, 1964; Ramalingam & Ravindranath, 1970), while the intine stained reddish-purple (Figs. 2B, 4A). The presence of phenolic residues in the exine was also suggested by observations of the autofluorescence of unstained sections. The exine fluoresced greenish-blue with ultraviolet excitation (Zeiss UG1 + FT420 filters) and yellow with blue excitation (Zeiss BG12 + FT510 filters). The exine also gave positive reactions with the protein-staining dyes Coomassie blue and amido black 10B (Fig. 4c), and a positive reaction with Sudan black B, indicating that lipids were present (Fig. 4D). The intine did not stain. The dye

5 Characteristics of poplar pollen walls Fig. i. Scanning electron micrographs of P. deltoides pollen. A, whole grain, x 2000; B, surface view of exine in detail, x Fig. 2. GMA-embedded sections of P. deltoides pollen, after suspension in mannitol- CaClj for 40 min, showing that few grains are structurally damaged by the treatment. Stained with toluidine blue ph 4-4. A, x 150; B, x 1100.

6 6 A. E. Ashford and R. B. Knox auramine O specifically caused the exine to fluoresce a bright yellow. The only other region giving a similar reaction was the stigma cuticle. Several methods gave good staining of the intine. The most striking result was obtained with Calcofluor white M2R new, when only the intine fluoresced a bright bluish-white colour (Fig. 5B). The intine was also PAS-positive (Fig. 4E) and metachromatic (red-purple) with toluidine blue (Fig. 4A). Treatment with pectinase removed most of the staining (Fig. 4B), but papain and hemicellulase were without effect. Micropores Exine 1 pm Intine Protoplast Fig. 3. Diagram interpreting wall structure in Populus. Both mature ungerminated pollen grains and grains germinating on the stigma surface reacted with aniline blue (Fig. 5C-F). However, many grains did not react, and in those that did the reaction was variable. In some grains there was a continuous deposit of aniline-blue-positive material around the entire circumference of the grain, associated with the intine (Fig. 5C-E). In others, fluorescent material occurred in isolated patches at the surface of the protoplast (Fig. 5D). There was consistently more material staining with aniline blue associated with the intine and protoplast of incompatible grains germinating on the stigma than in grains in compatible matings (compare Fig. 5 c with E). TO check whether the aniline-blue-positive material had Fig. 4. GMA-embedded sections of mature, ungerminated P. deltoides pollen (B) and P. deltoides pollen germinating on P. deltoides stigmas (A, C-F), stained with various reagents to illustrate the properties of the pollen wall. All photographs are magnified approx. x i indicates intine, e exine. A, stained toluidine blue, ph 4'4. The intine stains purple and exine green; B, section treated with pectinase for 12 h, then stained with toluidine blue, ph 44. Most of the intine material has been extracted while the exine is unchanged; c, stained with Coomassie blue; the exine and cytoplasm are stained but the intine is not; D, stained with Sudan black B. The exine and stigma cuticle are stained; E, stained with PAS reagents. Only the intine stains; F, stained for e3terase. Reaction occurs throughout the pollen cytoplasm and in the exine and also at the surface of the stigma (arrow).

7 Characteristics of poplar pollen walls

8 A F. Ashfnrd and R. B. KflOX

9 Characteristics of poplar pollen walls 9 been induced by fixation (Hughes, 1977) whole unfixed pollen (stored at 18 C) was mounted in the aniline blue solution and observed by fluorescence microscopy. About 50% of the pollen showed specific aniline blue-induced fluorescence in the outer region of the grain (Fig. 5F). Controls of both unfixed and fixed, sectioned material mounted in alkaline buffer displayed characteristic autofluorescence of the exine and protoplast which was much less intense than the aniline blue-induced fluorescence associated with the intine. The protein stains, Coomassie blue and amido black 10B mostly stained the cytoplasm in sections of glutaraldehyde-fixed, GMA-embedded material. There was no staining of the intine, although clearly some protein was associated with the exine (Fig. 4c). Pollen grains briefly fixed in cold glutaraldehyde and infiltrated and embedded at low temperature retained some hydrolytic enzyme activity. Esterase was present in cytoplasmic sites, and was also associated with the exine (Fig. 4F), while acid phosphatase had a similar cytoplasmic location but was restricted to the intine. Confirmation of the intine localization of acid phosphatase was obtained in fresh, nearly mature, pollen freeze-sectioned while still held in the anther. Immunofluorescence methods using rabbit antisera to pollen diffusates showed that antigens are located in both intine and exine sites. In anthers freeze-sectioned at the prevacuolate period of pollen development, the antigens were absent from the wall sites, activity being first evident in the intine during the vacuolate period. In pollen approaching maturity, but sectioned within the anther to avoid diffusion artifacts as far as possible, specific fluorescence was also associated with the exine cavities. Faint fluorescence was associated with the starch grains in the cytoplasm of mature pollen with both test sections treated with anti-pollen serum and control sections treated with normal serum. Origin of pollen diffusates in P. deltoides The patterns of protein release in Populus pollen grains have been followed using Coomassie blue stain-fixing media and cine-micrography. Analysis of 16-mm frames showed that the grains swelled in contact with the aqueous medium, and within 5 s protein emission from all around the pollen surface was detected as a purple halo. Fig. 5. GMA-embedded sections of mature ungerminated pollen (D), and pollen germinating on the stigma in compatible (P. deltoides x P. deltoides) matings (A, B, E), and incompatible (P. alba x P. deltoides) matings (c), stained with various fluorescent dyes and photographed using epi-fluorescence optics. The exine and cytoplasm are autofluorescent, but in all cases this is weaker than the specific dye staining, A, stained with auramine O. The exine is intensely fluorescent, x 1300; B,stained with Calcofluor M2R new. The intine fluoresces strongly, x 1300; c, incompatible mating, stained with aniline blue for callose. Note fluorescent material in the intine and in the cytoplasm, x 1300; D, ungerminated pollen stained with aniline blue. Note fluorescent material in the inner region of the intine of one grain and at the periphery of the cytoplasm in the other, x 1500; E, compatible mating stained with aniline blue. Fluorescent material occurs in the intine, x 1300; F, whole, unfixed pollen (stored > 12 months at 18 C) mounted in aniline blue and photographed directly. Fluorescence varies greatly in different grains, x CEL44

10 io A. E. Ashford and R. B. Knox Following this rapid reaction, there was a progressive release during the succeeding 2-5-3*0 min, of material stained dark blue from around the surface of the grain, presumably from the intine. Further evidence has been obtained using the pollen print technique in which dry pollen is placed on agarose films which are then stained with Coomassie blue to reveal the protein emission patterns. Within 5-30 s after contact, diffuse, particulars, stained material is present at the sites of the grains. After longer exposure the entire gel adjacent to the pollen is diffused with granular stained material. Characteristics of pollen diffusates Examination of pollen grains following treatment with 10% buffered mannitol and 10% CaCl 2 indicated that this treatment did not cause any obvious structural damage to the grains (Fig. 2 A, B). Results using the fluorochromatic test for membrane integrity showed that membranes were not leaky after the treatment. In fresh pollen samples, there was no significant difference between the proportion of grains accumulating fluorescein for fresh pollen and samples incubated for 40 min in 10% mannitol. Fig. 6. Drawings of protein and carbohydrate patterns in gels of P. deltoides and P. alba diffusates run on various electrophoretic systems. Protein bands were visualized by staining with amido black 10B. A, B, disk electrophoresis. Gel A shows P. deltoides diffusate protein pattern, with some 22 bands. Gel B is from the same run, stained with PAS reagents for carbohydrate: 2 PAS-positive bands correspond exactly with 2 major protein bands. C, D, protein patterns obtained on Gradipore continuous gradient gels of diffusate from P. deltoides (c) and P. alba (D) ; E, F, protein patterns from diffusates of P. deltoides (E) and P. alba (F) after dissociation in SDS and mercaptoethanol. Protein subunits from each diffusate fall into similar molecularweight classes.

11 Characteristics of poplar pollen walls 11 Lower concentrations of mannitol resulted in a 20% reduction of grains accumulating fluorescein, compared with fresh pollen. Fresh pollen of P. deltoides usuallyshowed a fiuorochromatic reaction in 88 % of the grains, while in stored pollen this dropped to 74%. Thus it was considered that the mannitol solution contained a pollen diffusate with little cytoplasmic contamination, rather than an extract. After ammonium sulphate precipitation and desalting, the diffusates ran well on a number of different polyacrylamide gel systems and, following staining with either amido black 10B or Coomassie blue, a large number of protein-staining bands were apparent. A typical run of P. deltoides diffusates in Tris-glycine buffer is shown in Fig. 6 A. Twenty-two protein bands were well resolved. There were slight variations in the number and position of bands in successive runs, acccording to the method of staining of the gels, and whether the diffusate was fresh or frozen. Staining of some gels with PAS reagents (Fig. 6 B) showed that some of the protein bands had associated carbohydrate. Two such bands were intensely stained and corresponded exactly with 2 major protein bands. A series of 3-4 other PAS-positive bands occurred closer to the origin. These were more faintly stained and were difficult to equate with particular protein bands. Cross-comparisons between banding patterns of P. deltoides and P. alba diffusates were difficult to make using disk electrophoresis because of slight variations in the gels both within and between successive runs. Comparisons were therefore made on the same gel, using Gradipore continuous gradient gels. There is a continuous decrease in pore diameter in these gels and so proteins are separated according to molecular size rather than electrophoretic mobility. The banding pattern of a typical run is shown in Fig. 6c, D. The method resolved fewer bands than disk electrophoresis: 16 bands were resolved for P. deltoides and 10 for P. alba. In P. alba there are 3 major regions where bands occurred, suggesting that proteins in 3 major molecular-weight classes were present. P. deltoides contained a corresponding group of low-molecular-weight proteins which ran close to the marker dye, and also a broad band of proteins of high molecular weight which partly overlapped a similar broad band in P. alba. The proteins in the middle-molecular-weight range did not appear to correspond. The actual molecular weights were not calculated from these gels. SDSgels After dissociation in SDS and mercaptoethanol, many bands were evident on electrophoresis (Fig. 6E, F). In P. deltoides, the major band towards the anode was of low mol. wt, c , while a group of bands towards the origin ranged in mol. wt up to The patterns for P. alba were very similar except that the lowmol.-wt fraction formed 2 clear diffuse bands of mol. wt c and , ranging to bands near the origin up to Isoenzyme patterns in pollen diffusates Pollen diffusates of both species were run on disk gels which were then stained for leucine aminopeptidase, acid phosphatase, esterase and peroxidase. These enzymes were all present in diffusates from both species (Figs. 7, 8).

12 12 A. E. Ashford and R. B. Knox Leucine aminopeptidase. Two isoenzymes of leucine aminopeptidase (R F 0-51 and o-6o) were found in P. deltoides diffusates (Fig. 7A), while only one (i? F 0-52) was present in P. alba (Fig. 7B). Thus the single P. alba isoenzyme had a similar electrophoretic mobility to one of the P. deltoides isoenzymes. u B = Fig. 7. Drawings of isoenzyme patterns in disk gels, A, B, leucine aminopeptidase from diffusates of P. deltoides (A) and P. alba (B). Both have one isoenzyme in common; C-E, acid phosphatase isoenzyme patterns from fresh P. deltoides pollen diffusates (c) for comparison with P. alba (E). D shows the isoenzyme pattern from the P. deltoides diffusate after freezing and thawing either the pollen or the diffusate. - Fig. 8. A, B, patterns of esterase isoenzymes in disk gels from P. deltoides (A) and P. alba (B) diffusates; c, D, peroxidase isoenzymes from P. deltoides (c) and P. alba (D) diffusates. Four peroxidase isoenzymes had similar electrophoretic mobilities. Add phosphatase. A total of 8 major isoenzymes of acid phosphatase occurred in fresh (unfrozen) diffusates of P. deltoides (Fig. 7 c). Two broad bands remained near the origin in the buffer system used. There were fewer acid phosphatase isoenzyme

13 Characteristics of poplar pollen walls 13 bands in P. alba (Fig. 7E). Five were well-stained and one stained faintly. The electrophoretic mobilities of 4 of the phosphatase bands were similar in the 2 species. Freezing and thawing of both P. deltoides pollen and their diffusates resulted in changes in the number and pattern of acid phosphatase bands (Fig. 7D). The 2 slowmoving bands, close to the origin, stained more faintly, while several additional fast-moving bands occurred close to the marker dye. It seems most likely that these represent derivatives of the enzyme bands close to the origin, and this suggests that, in spite of their extracellular location in the pollen grain, at least some of the isoenzymes of acid phosphatase are not stable to freeze-thawing. D D Fig. 9. Photograph of immunodiffusion gel of antigens from P. deltoides (wells D) and P. alba (wells A) diffusates, tested against anti-p. deltoides serum (anti D in the central well), showing precipitin bands. Esterase. Ten well-stained esterase bands were present in P. deltoides (Fig. 8A). P. alba also showed 10 well-stained bands (Fig. 8 B), but there were, in addition, 4 faintly stained bands, and the isoenzyme pattern was quite different in the two species. Patterns were the same whether the diffusates were of fresh or frozen pollen, or whether the diffusate used was itself fresh or frozen. Peroxidase. P. deltoides had 9 peroxidase isoenzymes (Fig. 8 c). Four showed similar electrophoretic mobility to those of P. alba. P. alba had fewer peroxidase isoenzymes than P. deltoides (Fig. 8D). There were only 3 major bands, and the 2 minor bands near the origin. The banding pattern in each case was unaffected by addition of 3-amino-i,2,4-triazole to the incubation medium, indicating that the reaction was due to peroxidase activity and not to catalase. Pollen-wall antigens and glycoproteins Pollen diffusates of P. deltoides tested against homologous antiserum gave 5 precipitin lines (Figs. 9, 10 A). Immunodiffusion tests against diffusate from P. alba revealed

14 14 A. E. Ashford and R. B. Knox that at least one antigen (third from the antigen well) is specific to P. deltoides pollen, while 2 are common with P. alba pollen (second and fifth from the antigen well). Pollen diffusates of P. alba, when tested against anti-p. alba, gave 6 precipitin lines, 2 being specific, 2 showing partial identity and 2 being common with P. deltoides (Fig. IOB). Immunoelectrophoresis of P. deltoides diffusates against homologous antiserum gave 7 precipitin lines, the 2 major arcs being cathodic, the others migrating towards the anode (Fig. 10 c). D O anti D anti A Fig. 10. A, B, Drawings of precipitin patterns from immuno diffusion plates of P. deltoides antigens (well D) and P. alba antigens (well A) from pollen diffusates, diffused against anti-p. deltoides serum (anti D) and anti-p. alba serum (anti A). c, immunoelectrophoresis pattern of antigens from P. deltoides diffusate (in well D) electrophoresed against anti-p. deltoides serum (anti D) placed in the rectangular well. The presence of glycoproteins or polysaccharides in the pollen diffusates was further indicated by interaction with the lectin Concanavalin A. Double diffusion in agarose gels resulted in multiple precipitin bands, which were abolished by incubation of the pollen diffusate with methyl a-d-mannoside. DISCUSSION In this paper we have used a number of methods to characterize the pollen diffusates shown previously to be important in the recognition reactions on the stigma that control interspecific incompatibility between black and white poplars (Knox et al. 1972). Several tests have indicated that these are diffusates that are not likely to have

15 Characteristics of poplar pollen walls 15 been contaminated significantly by cytoplasmic constituents. They have been shown to be remarkably complex, using a range of different protein analytical techniques. Some 22 protein bands of differing electrophoretic mobility were detected with polyacrylamide gel disk electrophoresis, while the continuous gradient pore gels showed that these proteins were of widely differing molecular weight. The major fractions in both pollen types have molecular weights greater than and presumably are not denatured. After partial denaturation in SDS gels, a large number of bands was obtained in P. deltoides, ranging in molecular weight between and , indicating a great diversity in subunit structure. The patterns obtained for the SDS gels of Populus are remarkably similar to those found previously in Cosmos and Ambrosia pollen diffusates (Howlett, Knox, Paxton & Heslop-Harrison, 1975). At least 5 of the protein bands had carbohydrate associated with them and so are presumed to be glycoproteins. This was indicated both by their PAS reaction in gels, and their binding to Con A. Glycoproteins have also been detected in the pollen walls of grasses, especially Zea mays, by their binding to Con A (Watson, Knox & Creaser, 1974). The discovery of glycoproteins in pollen wall sites and diffusates in several different types of pollen grain is a significant finding. The recognition potential of glycoproteins far exceeds that of proteins alone and plant glycoproteins have been implicated widely in recognition reactions in plants (Clarke & Knox, 1978). These might be the compounds involved in recognition reactions at the stigma surface of Populus. The pollen diffusates also contained a range of enzymes. This is to be expected: some 12 enzymes are known to be stored in the walls of pollen grains, largely from cytochemical studies (Knox, Heslop-Harrison & Heslop-Harrison, 1975). We now add to this list peroxidase and the dipeptidase, leucine aminopeptidase, and confirm the presence of esterase and acid phosphatase. The cytochemical studies with the low-temperature-embedding technique show that acid phosphatase is present in the intine and esterase in the exine in the poplar pollen grain, as has been established for other pollen grains using freeze-sectioning. With the exception of leucine aminopeptidase, all enzymes showed a complex isoenzyme pattern in both poplar species investigated. Some of the isoenzymes were common to both species, in contrast with the total protein patterns. The isoenzyme patterns contrast with those of Howlett et al. (1975) which show only 2 isoenzymes of esterase and 6 of acid phosphatase in diffusates of Cosmos bipinnatus pollen. Other authors have reported a high degree of polymorphism of esterase isoenzyme in extracts from several pollen grain types (see review by Brewbaker, 1971). However, the exact site of origin of these esterases is not known, and some of them may have been of cytoplasmic origin. This is indeed likely since many of the isoenzymes had similar electrophoretic mobilities to those extracted from other tissues in the same plant. Cytochemical staining reactions of the exine and intine indicate that the composition of these wall layers is typical (Heslop-Harrison, 1975). The retention of acid phosphatase and esterase activities in the intine and exine, respectively, in low-temperatureembedded pollen suggests that in spite of the massive efflux of proteins and glycoproteins from the grains on wetting, some protein does remain behind in the wall.

16 16 A.E. Ashford and R. B. Knox Failure of the protein-staining dyes Coomassie blue and amido black 10B to give a positive reaction with the intine suggests that these amounts are small. The aniline blue-positive material in the pollen was most probably a /?-i,3-glucan, such as callose, or a mixed /ff-1,3-, /?-i,4-glucan (see Herth et al. 1974; Vithanage & Knox, 1977; Smith & McCully, 1978). The occurrence of /?-i,3-glucans in the cytoplasm and intine of mature ungerminated pollen was an unexpected result. It is well established that callose is produced in the stigma in the vicinity of incompatible pollen tubes, and this is considered to be a feature diagnostic of a rejection response in sporophytic self-incompatibility systems (Heslop-Harrison, 1975). Similarly, callose occlusion of incompatible pollen tubes is a widespread phenomenon and in some species callose deposition also occurs in the pollen grain in incompatible matings (Shivanna, Heslop-Harrison & Heslop-Harrison, 1978). Callose deposition in pollen tubes in the wall and in the cytoplasm to form callose plugs is also a normal feature of compatible matings (Cresti & Van Went, 1976). In poplar, it is clear that there is increased deposition of callose in the pollen tubes and grains in incompatible matings (Knox et al. 1972). We also have evidence of a callose rejection response in poplar stigmas (A. E. Ashford & R. B. Knox, in preparation). However, the presence of /?-i,3-glucans in ungerminated grains and in germinating P. deltoides grains and their pollen tubes in compatible matings suggests that the capacity for /?-i,3-glucan production already exists in the pollen and that the callose response of poplar pollen in incompatible matings is merely a matter of degree. This work was begun while both authors were at the Botany Department, Australian National University, Canberra, and has been supported by grants from the Australian Research Grants Committee, including a research fellowship to A.E.A. We thank Mr R. R. Willing for provision of plant material, Dr B. K. Filshie of CSIRO Division of Entomology for valued help with scanning electron microscopy, and Dr J. V. Jacobsen for preparation of SDS gels. We are grateful for assistance from Miss K. Gee in the preparation of some of the diagrams and Mr L. Meier for photography. A gift of calcofluor white M2R from the American Cyanamid Company is also gratefully acknowledged. REFERENCES ASHFORD, A. E., ALLAWAY, W. G. & MCCULLY, M. E. (1972). Low temperature embedding in glycol methacrylate for enzyme histochemistry in plant and animal tissues. J. Histochem. Cytochem. 20, BARKA, T. & ANDERSON, P. J. (1962). Histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler. J. Histochem. Cytochem. 10, BREWBAKER, J. L. (1971). Pollen enzymes and isoenzymes. In Pollen Development and Physiology (ed. J. Heslop-Harrison), pp London: Butterworth. CLARKE, A. E. & KNOX, R. B. (1978). Cell recognition in flowering plants. Q. Rev. Biol. 53, CRESTI, M. & VAN WENT, J. L. (1976). Callose deposition and plug formation in Petunia pollen tubes in situ. Planta 133, DAVIS, B. J. (1964). Disc electrophoresis. II. Methods and applications to human serum protein. Ann. N.Y. Acad. Set. 121, ERDTMAN, G. (1966). Pollen Morphology and Plant Taxonomy, Vol. I. Angiosperms, 2nd edn. New York: Hafner. FEDER, N. & O'BRIEN, T. P. (1968). Plant microtechnique: some principles and new methods. Am. J. Bot. 55,

17 Characteristics of poplar pollen walb FISHER, D. B. (1968). Protein staining of ribboned epon sections for light microscopy. Histochemie 16, HERTH, W., FRANKE, W. W., BITTIGER, H., KUPPEL, A. & KELLICH, G. (1974). Alkali-resistant fibrils of fi-1,3- and /?-i,4-glucans; structural polysaccharides in the pollen tube wall of Lilium longiflorum. Cytobiologie 9, HESLOP-HARRISON, J. (1975). Incompatibility and the pollen-stigma interaction. A. Rev. PL Physiol. 26, HESLOP-HARRISON, J. & HESLOP-HARRISON, Y. (1970). Evaluation of pollen viability by enzymatically induced fluorescence; intracellular hydrolysis of fluorescein diacetate. Stain Technol. 45, HESLOP-HARRISON, J., KNOX, R. B. & HESLOP-HARRISON, Y. (1974). Pollen wall proteins: exine-held fractions associated with the incompatibility response in Cruciferae. Theor. appl. Genet. 44, HESLOP-HARRISON, Y. (1977). The pollen-stigma interaction: pollen-tube penetration in Crocus. Ann. Bot. 41, HOWLETT, B. J., KNOX, R. B., PAXTON, J. D. & HESLOP-HARRISON, J. (1975). Pollen-wall proteins: physicochemical characterization and role in self-incompatibility in Cosmos bipinnatus. Proc. R. Soc. B 188, HUGHES, J. E. (1977). Aspects of infrastructure and Function in Arbutilon Nectaries. M.Sc. thesis, Australian National University. HUGHES, J. & MCCULLY, M. E. (1975). The use of an optical brightener in the study of plant structure. Stain Technol. 50, KNOX, R. B. (1970). Freeze sectioning of plant tissues. Stain Technol. 45, KNOX, R. B. (1971). Pollen-wall proteins: localization, enzymic and antigenic activity during development in Gladiolus (Iridaceae). J. Cell Sci. 9, KNOX, R. B., HESLOP-HARRISON, J. & HESLOP-HARRISON, Y. (1975). Pollen-wall proteins. In The Biology of the Male Gamete (ed. J. G. Duckett & P. A. Racey), pp London: Academic Press. KNOX, R. B., WILLING, R. R. & ASHFORD, A. E. (1972). Role of pollen-wall proteins as recognition substances in interspecific incompatibility in poplars. Nature, Lond. 237, KNOX, R. B., WILLING, R. R. & PRYOR, L. D. (1972). Interspecific hybridisation in poplars using recognition pollen. Silvae Genet. 21, MAEDA, H. & ISHIDA, N. (1967). Specificity of binding of hexopyranosyl polysaccharides with fluorescent brightener. J. Biochem., Tokyo 62, NACHLAS, M. N., CRAWFORD, D. T. & SELIGMAN, A. M. (1957). The histochemical demonstration of leucine aminopeptidase. J. Histochem. Cytochem. 5, NOVIKOFF, A. B. & GOLDFISCHER, S. (1969). Visualisation of peroxisomes (microbodies) and mitochondria with diaminobenzidine. J. Histochem. Cytochem. 17, O'BRIEN, T. P., FEDER, N. & MCCULLY, M. E. (1964). Polychromatic staining of plant cell walls by toluidine blue O. Protoplasma 59, PEARSE, A. G. E. (1968). Histochemistry, Theoretical and Applied, vol. 1, 3rd edn. London: Churchill. PEARSE, A. G. E. (1972). Histochemistry, Theoretical and Applied, vol. 2, 3rd edn. Edinburgh and London: Churchill-Livingstone. RAMALINGAM, K. & RAVINDRANATH, M. H. (1970). Histochemical significance of green metachromasia to toluidine blue. Histochemie 24, ROWLEY, J. R. & ERDTMAN, G. (1967). Sporoderm in Populus and Salix. Grana polynol. 7, SHIVANNA, K. R., HESLOP-HARRISON, Y. & HESLOP-HARRISON, J. S. (1978). Inhibition of the pollen tube in the self-incompatibility response of grasses. Incompatibility Newsletter 10, 5-7. SMITH, M. M. & MCCULLY, M. E. (1978). A critical evaluation of the specificity of aniline blue induced fluorescence. Protoplasma 95, VITHANAGE, H. I. M. V. & KNOX, R. B. (1977). Callose: its nature and involvement in selfincompatibility responses in sunflower, Helianthus armuus, and the grasses, Lolium perenne and Secale cereale. Incompatibility Newsletter 8, WATSON, L., KNOX, R. B. & CREASER, E. H. (1974). Con A differentiates among grass pollens by binding specifically to wall glycoproteins and carbohydrates. Nature, Lond. 249, iy (Received 5 December 1979)

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