POLLEN-WALL PROTEINS: QUANTITATIVE CYTOCHEMISTRY OF THE ORIGINS OF INTINE AND EXINE ENZYMES IN BRAS SIC A OLERACEA

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1 J. Cell Sci. 21, (1976) 423 Printed in Great Britain POLLEN-WALL PROTEINS: QUANTITATIVE CYTOCHEMISTRY OF THE ORIGINS OF INTINE AND EXINE ENZYMES IN BRAS SIC A OLERACEA H. I. M. V. VITHANAGE AND R. B. KNOX School of Botany, University of Melbourne, Parkville 3052 Victoria, Australia SUMMARY Simultaneous coupling methods for detection of acid phosphatase and non-specific esterase produce a coloured reaction product that is quantitatively related to enzyme content in freezesectioned Brassica pollen and tapetal cells. The intine-located acid phosphatase has 2 periods of synthesis: the first in late vacuolate period, associated with the completion of deposition of the intine polysaccharides; the second during pollen maturation, apparently reflecting cytoplasmic synthesis. Esterase activity accumulates in the tapetal cells until dissolution at early maturation period, when there is a dramatic rise in pollen-wall esterase activity, reflecting the transfer from tapetum to exine cavities. These quantitative studies confirm the gametophytic and sporophytic origins of the intine and exine proteins. INTRODUCTION In Brassica oleracea var. acephala, marrow-stem kale, as in many other Cruciferae, self-incompatibility is controlled by a single major genetic locus, S, with many alleles, and the behaviour of the haploid pollen grains on the stigma is determined by the genotype of its diploid parent (Heslop-Harrison, 1967; Stout, 1931; Thompson, 1957; Thompson & Howard, 1959). Such sporophytic self-incompatibility systems are common in the Cruciferae and Compositae, but the physiological mechanism for such behaviour has only recently been elucidated. Heslop-Harrison (1968) predicted from studies of pollen exine development that the active factors controlling pollen behaviour were likely to be products of parental synthesis deposited on the pollen during its development within the anther. This hypothesis has been confirmed, first with the finding that pollen-wall proteins occur in the outer cavities of the exine, whose site of synthesis is the diploid tapetal cells, as demonstrated by transmission electron microscopy of pollen development in Malvaceae (Heslop-Harrison, Heslop-Harrison, Knox & Howlett, 1973), and Cruciferae (Dickinson & Lewis, 1973 a, b). Light-microscope cytochemistry has demonstrated the transfer of proteins from tapetum to pollen exine cavities in Iberis and other Cruciferae (Knox, Howlett, Heslop-Harrison & Heslop-Harrison, 1973; Heslop-Harrison, Knox & Heslop- Harrison, 1974). Secondly, it has been shown that the exine-borne fraction is active

2 424 H. I. M. V. Vithanage and R. B. Knox in inducing the rejection response of self incompatibility in Cruciferae (Dickinson & Lewis, 19736; Knox et al. 1973; Heslop-Harrison et al. 1974). The exine proteins are the first to be released when pollen alights on the stigma, or is moistened (Heslop-Harrison et al. 1973; see Heslop-Harrison, 1975 a, b). They comprise a heterogeneous group of proteins, together with lipids, pigments and monosaccharides. In Brassica, 6 protein fractions have been detected in exinederived extracts, ranging in molecular mass from to (Knox, Heslop- Harrison & Heslop-Harrison, 1975). Characteristic enzyme markers include nonspecific esterase. In contrast, the inner intine wall layer contains enzymic and antigenic proteins incorporated within its polysaccharide matrix (Knox & Heslop-Harrison, 1970a). These are generally released more slowly during pollen germination and are associated with the developing pollen tube, which may bear a plug of hydrolytic enzymes at its tip. These enzymes are products of gametophytic synthesis, and characteristic markers include acid phosphatase and ribonuclease (Knox et al. 1975). This paper describes attempts to use quantitive cytochemical methods to demonstrate the sporophytic and gametophytic origins of the exine and intine proteins. The development of 2 marker enzymes: esterase, characteristically located in the exine cavities, and acid phosphatase, found in the intine have been followed. MATERIALS AND METHODS Plants of Brassica oleracea var. acephala, kale, of genotype S 8 S 8) were grown from seed obtained from the Plant Breeding Institute, Cambridge, U.K., under controlled conditions with natural illumination in short days of 8 h light at 12/10 C day/night temperatures. The anthers of these plants were used in the present investigation. Identification of developmental stages The fluorochromatic reaction was used to test pollen viability (Heslop-Harrison & Heslop- Harrison, 1970), and to classify developmental stages of Brassica pollen grains, essentially as described by Knox & Heslop-Harrison (19706) and Knox (1971). Tissue preparation Anthers were prepared for sectioning by the method of Knox (1970). Petri dishes containing 15 % (w/v) gelatin and 2% (v/v) glycerol were prepared as embedment and used for freeze-sectioning at i5 C. Sections of thickness 2 /(m or greater were cut and placed on microscope slides pre-coated with 1 % gelatin and air-dried. These slides were stored at 4 C for short periods until used in cytochemical tests. Polysaccharide localization The periodic acid-schiff (PAS) reaction (Pearse, 1972) was used to detect pollen polysaccharides containing vicinal glycol groups. Intine deposition can be followed with this method. Rapid discrimination between exine and intine was made by staining sections in toluidine blue O (Jacobsen, Knox & Pyliotis, 1971). Sections were stained for 2 min, mounted in water, and examined. The intine stained red, while the exine was brilliant green.

3 Origins of inline and exine enzymes 425 Quantitative enzyme cytochemistry Acid phosphatase. This enzyme was localized in the anther sections using the method of Barka & Anderson (1962), as modified by Knox & Heslop-Harrison (1970a). The anther sections were incubated for 10 min in a solution of a-naphthyl acid phosphate (1 mg/ml) and hexazorized pararosanilin (2 mg/ml) buffered at ph 6-0. The reaction was stopped by rinsing the slides in distilled water. The slides were then dried and mounted in Eukitt (Kindler, Freiburg, Germany). Control sections were made by omitting the substrate in the reaction mixture. For quantitative studies, batches of slides were treated under identical conditions Q 0-6 O Section thickness, ftm Fig. 1. Quantitative relationship between section thickness and reaction product for esterase in freeze-sectioned pollen (closed squares) and tapetal cells (open squares). Standard errors of the mean for 16 measurements are indicated. Non-specific esterases. These were localized in the anther section using the method given by Pearse (1972), using the substrate a-naphthyl acetate coupled to hexazotized pararosanilin. Quantitative estimation of enzyme activity. All measurements were made using a Carl Zeiss SMP-05 Scanning Microscope Photometer. The absorption maxima of reaction products for esterase and acid phosphatase were at 520 and 490 nm respectively and all subsequent measurements were made at these wavelengths. Readings were made of cores of tissue within both tapetal cells and pollen grains in anther sections 2 fim thick using a core of 1CV4 /tm diameter. Sections from anthers within the same block were used, 4 measurements being made within pollen grains and 4 within tapetal cells of each, making a total of 16 measurements for both pollen and tapetum at each developmental period. Similar measurements of optical density per unit core volume were recorded for pollen and tapetum in test and control sections for parallel sections stained for esterase and acid phosphatase. All the values given for enzyme activity are corrected by subtracting optical densities of controls. The variation of the control values reflects the ability of the pollen walls to bind the dye non-specifically during development. To determine that the cytochemical reactions for both enzymes are quantitative under the

4 426 H. I. M. V. Vithanage and R. B. Knox conditions used, and follow Beer's Law, anthers 7 mm in length were sectioned at thicknesses ranging from 2 to 20 /(m and reacted for esterase under the standard conditions. The optical densities obtained for both pollen and tapetum were plotted against section thickness. Fig. 1 shows the values fit a regression line: Pollen: y=o - i36 + o-o4sa; Tapetum: Y = F= *** F=45-25O*** These values indicate that a significant portion of the variance of optical density is explained by the regression, demonstrating the quantitative nature of the reaction product detected. As a test of the accuracy of section thickness in the developmental series, parenchyma cells adjacent to the anther vascular tissue were selected as a control area showing little change during development. Enzyme activity was measured to detect any marked departures that might suggest fluctuations in section thickness. Student's t test applied to the data showed a very high level of significance, P < 0001, indicating that no significant departures were encountered. OBSERVATIONS Qualitative cytochemistry of pollen-wall enzymes Meiosis. This period occurred in buds up to 2-5 mm in length. Pollen mother cells showed absence of enzyme activity, both for acid phosphatase and esterase. Faint staining was detected in the tapetal cells for both enzymes. However, at tetrad period in buds 3 mm in length, the microspore cytoplasm showed some esterase activity, with a denser reaction product than in the surrounding tapetal cells. Pre-vacuolate period. This period was found in buds 4 mm in length. The tapetal cells showed diffuse esterase activity, but only faint staining for acid phosphatase (Fig. 4 A). The developing exine of the young microspores showed considerable nonspecific absorption of the coupling agent, appearing a dark yellow colour in control sections. No enzyme activity was detected for either marker, and deposition of the intine polysaccharides had not commenced. Vacuolate period. The early vacuolate period, characterized by the onset of vacuolation in the pollen cytoplasm, was characteristic of buds 4-5 mm in length. Tapetal cells were highly vacuolated, and while showing only faint acid phosphatase activity (Fig. 2 A), had accumulated considerable esterase activity, diffuse throughout the cells (Fig. 3 A). The pollen grains showed acid phosphatase activity associated with the intine only at the 3 germinal apertures (Fig. 4B). Intine polysaccharide synthesis is initially concentrated at these sites, as seen from sections treated with the PAS reaction (Fig. 4c). No esterase activity was associated with the pollen walls but the cytoplasm was moderately stained (Fig. 3 A). Mid-vacuolate pollen grains, characterized by a large central vacuole, peripheral cytoplasm, and single nucleus were found in buds 5-0 mm in length. Tapetal cells showed accumulation of acid phosphatase, especially at the periphery of the cytoplasm on the inner side adjacent to the pollen grains (Figs. 2B, 4D). Moderately dense deposition of esterase reaction product occurred throughout these cells, associated with spherical granules 2 /(m in diameter in the highly vacuolated cytoplasm (Figs. 3B, 5 A). Pollen grains showed dense reaction product for acid phosphatase associated with the intine layer all around the grains (Figs. 2C, 4D), corresponding

5 Origins of intine and exine enzymes 4*7 Fig. 2. Freeze-sectioned anthers of Brassica oleracea showing localization of acid phosphatase during development. A, early vacuolate period; B, mid-vacuolate period; c, late vacuolate period; D, early maturation period; E, late maturation period; F, control section incubated in absence of substrate, x 300. Fig. 3. Freeze-sectioned anthers showing localization of non-specific esterase during development, A-F: parallel sections to those used in Fig. 2. x 1100.

6 H. I. M. V. Vithanage and R. B. Knox 4 A : B C v<

7 Origins of inline and exine enzymes 429 to the intine polysaccharides revealed by PAS reaction (Fig. 4E). Some slight esterase activity was detected in the pollen, associated with the peripheral cytoplasm (Figs. 2B, 5 A). The late vacuolate period, characterized by cytoplasmic synthesis and consequent change in appearance of the central vacuole, which is often replaced by several smaller vacuoles, occurred in buds 7 mm in length. Tapetal cells were extensively vacuolated and enlarged in radial thickness, and showed increased acid phosphatase activity (Figs. 2 c, 4F), especially at the inner margins. Very dense reaction product for esterase was obtained (Figs. 3 c, 5B), the spherical granules being very much enlarged - up to 5 fim in diameter - and apparently in clusters within a larger structure, possibly the vacuoles described by Dickinson & Lewis (19736). Distribution of lipids (in elaioplasts) shows an entirely different pattern (Fig. 5 E). Pollen grains showed dense deposition of reaction product for acid phosphatase in the intine, especially clear in equatorial sections (Figs. 2 c, 4F). Activity is present in the intine plugs at the germinal apertures (Fig. 4F, G), and around the interior of the exine, but is absent from the exine cavities. In contrast, esterase is present at moderate activity in the cytoplasm, and is absent from the pollen walls (Figs. 3 c, 5B). Maturation period. Early maturation period is characterized by binucleate pollen grains, increased cytoplasmic synthesis with great reduction in vacuoles, and occasional starch grains in buds 8-5 mm in length. Tapetal cells were undergoing dissolution and showed reduction in radial thickness, and great irregularity in cell shape; intense esterase activity was detected (Figs. 3D, 5c) but only slight phosphatase activity (Fig. 2D). Pollen grains, still generally round, have intense phosphatase activity in the intine, and moderate activity in the cytoplasm filling the grains was detected. Esterase activity was still present in the cytoplasm but a thin line of reaction product was detected associated with the intine at the germinal apertures, on the inner side. Later stages of maturation are characterized by a change in shape of the pollen, which becomes markedly trilobed in equatorial section (Figs. 4H, 41), and trinucleate, with dense cytoplasm filled with starch grains in buds 10 mm in length. Fig. 4. Freeze-sectioned pollen showing acid phosphatase or polysaccharide localization at various development periods, A, pre-vacuolate period section treated for acid phosphatase, showing absence of activity; B, early vacuolate period showing slight enzyme activity associated with the intine; C, parallel section to that of B, treated with PAS reaction for polysaccharide localization, showing some reaction associated with poral intine, and non-specific staining of exine also present in control section; D, mid-vacuolate period showing acid phosphatase activity associated with differentiating intine, and cytoplasmic activity in tapetal cells at top of picture (x 1100); E, parallel section to D treated with PAS reaction showing the presence of intine polysaccharides (xnoo); F, late vacuolate period showing intense acid phosphatase activity associated with the intine, seen in surface view in some grains, the pollen cytoplasm showing little activity (xnoo); c as F showing detail of individual pollen grain (x 1900); H-I, maturation period: H shows acid phosphatase activity in intine, conspicuous at germinal pores, and in cytoplasm, 1 shows PAS reaction for polysaccharides (x 1100). 2S-2

8 H. I. M. V. Vithanage and R. B. Knox B Fig. 5. Freeze-sectioned tapetal cells at various stages of development showing localization of esterase activity, A, mid-vacuolate period; B, late-vacuolate period; C, early maturation; D, late maturation, showing pollen grains after enzyme transfer; E, parallel section to B showing localization of lipids with oil red O (Pearse, 1968) for comparison with esterase activity, x The tapetum is absent, having undergone dissolution (Figs. 2E, 3E). Pollen grains show dense reactions for acid phosphatase in the intine and in the cytoplasm (Fig. 4H). Reaction for esterase is very dense in the surface exine cavities, and moderate in the cytoplasm (Fig. 3E, 5D).

9 Origins of inline and exine enzymes 431 Quantitative changes in enzyme activity during anther development The striking changes in enzyme activity and distribution described above have been estimated using quantitative microscopic methods. Cores of diameter 10-4 /im were measured within the tapetal cells and pollen grains at the various stages of development. The results, shown in Fig. 6, precisely quantitate the qualitative observations, reflecting the major syntheses and activities of the 2 marker enzymes during anther development. E _. 0 o * 10 q O Bud length, mm Fig. 6. Quantitative estimation of enzyme activities during anther development in Brassica oleracea. A shows increase in pollen grain size; B shows acid phosphatase activity in pollen (A) and tapetal cells (A); c shows esterase activity in parallel sections to those of B in pollen ( ) and tapetal cells ( ). Standard errors of the mean are indicated where they exceed the size of the symbol.

10 432 H. I. M. V. Vithanage and R. B. Knox During development, the pollen grains increased in diameter from 17 to 32 /mi. This increase is nearly linear, and since standard volume measurements were made of the pollen grains, the increase in pollen size means that total enzyme activity per pollen grain is underestimated at the later stages of development. Corrected values for enzyme activities expressed on a per pollen grain basis are shown in Table 1. These reveal that for acid phosphatase in the pollen grains, there are 2 periods of enzyme accumulation or synthesis, the first culminating in the late vacuolate period Table 1. Enzyme activity of pollen grains at various stages of development, corrected to account for increase in pollen volume Period Pre-vacuolate Early vacuolate Mid-vacuolate Late vacuolate Early maturation Late maturation Mature pollen Bud length, mm S 5' Pollen grain Core vol Pollen grain diam., /Mil i i'5 Enzyme activity per pollen grain* 1 Acid phosphatase, Esterase, O.D./ O.D./ unit vol. unit vol. ice S and corresponding to the deposition of the intine polysaccharides; and the second at the time of pollen maturation, corresponding to cytoplasmic synthesis. Between these 2 maxima there is a marked decline in activity early in the maturation period, which could be associated with intine storage mechanisms (see Discussion). The pattern for esterase is quite distinct, and shows that there is very little pollen-associated enzyme activity until late maturation period, when transfer of the exine proteins from the tapetal cells occurs (see Discussion). Fig. 6 shows the comparison between tapetal and pollen-associated enzyme activities during anther development. These values represent comparative absorbance units from cores of similar volume in the developing tapetal cells and pollen grains. The 2 peaks of acid phosphatase accumulation or synthesis in the pollen grains are evident, while the associated tapetal cells showed a gradual rise then fall in tapetal enzyme activity. The results for esterase activity are quite different. Tapetal cells show a dramatic increase in enzyme activity, commencing at spore release period in buds of 4 mm in length, and rising almost linearly to a maximum at early maturation period, just prior to tapetal dissolution. Pollen-associated esterase is low until this period, when a dramatic rise in activity occurred, reaching a maximum in mature pollen.

11 Origins of inline and exine enzymes 433 DISCUSSION Esterase, a marker enzyme known to occur in pollen exines of many plants including Brassica oleracea (Knox et al. 1975), has been shown by other methods to be synthesized in the diploid tapetal cells and thus to have a sporophytic origin. Our work shows that synthesis commences at spore release period and reaches a maximum shortly before dissolution early in pollen maturation period. During this time, pollen-associated esterase activity is at a low level. Early in maturation period, after the pollen grain mitosis, however, at exactly the time that tapetal dissolution is occurring, massive accumulation of enzyme activity occurs on the pollen surfaces, filling the exine cavities. The amount of pollen-associated enzyme activity appears close to that released from the tapetal cells. Such quantitative data add objective weight to the elegant observations of Dickinson & Lewis (19730,6) and Heslop-Harrison et al. (1973, 1974) who used electron microscopy and qualitative cytochemical methods to demonstrate a transfer of proteins and lipids in various Cruciferae and Malvaceae. As noted, esterase activity is to be looked upon as a marker for this transfer; it is apparent from other evidence that appreciable amounts of non-enzymic protein are likely also to be conveyed. In Brassica, enzyme activity in the freeze-sectioned material is associated with spherical granules about 2 /mi in diameter, usually contained singly or in clusters within larger organelles, probably the vacuoles described in transmission electron micrographs by Dickinson & Lewis (19736, fig. 13). The constancy of size of the Brassica granules is striking, the larger diameters consisting of clusters of 2-/tm vesicles. The parallels between such structures, and the plant 'lysosomes' described by recent authors, is interesting (for example, maize root hydrolases: Matile, 1968; Ashford & McCully, 1970; Parish, 1975). Zymograms of pollen esterases show the polymorphic nature of these enzymes. Brewbaker (1971) presented patterns for esterases of Zea mays and Hemerocallis aurantiaca, both showing several groups of bands. The Hemerocallis data included patterns from anther and style tissue, which revealed that pollen samples contained only two unique bands, the other 14 bands also occurring within the anther (presumably including the tapetum) and style. Most of the esterase synthesis would thus appear to be under sporophytic control. These esterases showed a broad spectrum of activity on a variety of organic substrates. Acid phosphatase has been shown to be a characteristic marker enzyme for the gametophytic intine proteins (Knox et al. 1975). This is further supported by the present evidence, the 2 major peaks of synthesis or accumulation during pollen development being apparently unrelated to events in the adjacent tapetal cells. This enzyme has been previously studied quantitatively during anther development in Lilium henryi (Linskens, 1966). Both pollen grains and tapetum showed a single peak of activity, with maxima midway during pollen development. The enzyme is, like esterase, polymorphic, and two or more isozymes have been detected in mature pollen and anthers of several plants (Linskens, 1966; Brewbaker, 1972; Howlett et al.

12 434 H -! M - v - Vithanage and R. B. Knox 1973; Knox & Ashford, in preparation). It will be of interest to determine whether the two peaks of activity involve different isozymes. The reduction in enzyme activity in pollen at the end of the vacuolate period, following completion of intine deposition, is of considerable interest. This occurs at a time when the anther cavity is about to commence dehydration as a prelude to release of mature dry pollen. A possible explanation for the loss of enzyme activity could be that it is associated with the mechanism of storage within the polysaccharide matrix; it is known from other systems that enzyme activity may diminish during ' packaging', presumably because of conformational changes. There seems little doubt that the final peak of activity reflects synthetic activity within the pollen cytoplasm. Work is in progress to follow the activity of these enzymes in other pollens with sporophytic control of incompatibility, including Helianthus annuus and Cosmos bipinnatus, where patterns similar to those now detected in Brassica may be expected. Of even greater interest, however, will be the results from studies of pollens with gametophytic control of pollen-stigma reactions, as in grasses such as Lolium perenne, where the intine proteins are thought to play a major role in recognition (see Heslop- Harrison, 19756). We are grateful to Professor J. Heslop-Harrison, F.R.S., Dr Adrienne Clarke and Mr J. Considine for helpful discussion of these results, and to the Australian Research Grants Committee for financial support (to R.B.K.). The work was carried out while one of us (H.I.M.V.V.) was supported by an Australian Government Commonwealth Post-graduate Research Award. We thank Mr C. Aeberli for growing the experimental plants. REFERENCES ASHFORD, A. E. & MCCULLY, M. E. (1970). Localization of naphthol AS-BI phosphatase activity in lateral and main root meristems of pea and corn. Protoplasma 70, 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: Butterworths. DICKINSON, H. G. & LEWIS, D. (1973a). Cytochemical and ultrastructural differences between intraspecific compatible and incompatible pollinations in Raphanus. Proc. R. Soc. B 183, DICKINSON, H. G. & LEWIS, D. (19736). The formation of the tryphine coating the pollen grains of Raphanus and its properties relating to the self-incompatibility system. Proc. R. Soc. B 184, HESLOP-HARRISON, J. (1967). Ribosome sites and S gene action. Nature, Lond. 218, HESLOP-HARRISON, J. (1968). Tapetal origin of pollen coat substances in Lilium. New Phytol HESLOP-HARRISON, J. (1975 a). Incompatibility and the pollen-stigma interaction. A. Rev. PI. Physiol. 26, HESLOP-HARRISON, J. (19756). The physiology of the incompatibility reaction in the Cruciferae. Proc. Eucarpia meeting, Scottish Horticultural Res. Inst., pp Edinburgh: Oliver & Boyd. HESLOP-HARRISON, J. & HESLOP-HARRISON, Y. (1970). Evaluation of pollen viability by enzymatically induced fluorescence; intracellular hydrolysis of fluorescein diacetate. Stain Teclmol. 45, HESLOP-HARRISON, J., HESLOP-HARRISON, Y., KNOX, R. B. & HOWLETT, B. (1973). Pollen-wall proteins: 'gametophytic' and 'sporophytic' fractions in the pollen walls of the Malvaceae. Ann. Bot. 37,

13 Origins of inline and exine enzymes 435 HESLOP-HARRISON, J., KNOX, R. B. & HESLOP-HARRISON, Y. (1974). Pollen-wall proteins: exine-held fractions associated with the incompatibility response in Cruciferae. Theoret. appl. Genet. 44, HESLOP-HARRISON, J., KNOX, R. B., HESLOP-HARRISON, Y. & MATTSSON, O. (1975). Pollenwall proteins: emission and role in incompatibility responses. In The Biology of the Male Gamete (ed. J. G. Duckett & P. A. Racey), Biol.J. Linn. Soc. 7, Suppl. i, JACOBSEN, J. V., KNOX, R. B. & PYLIOTIS, N. A. (1971). The structure and composition of aleurone grains in the barley aleurone layer. Planta 101, HOWLETT, B. J., KNOX, R. B., PAXTON, J. D. & HESLOP-HARHISON, J. (1975). Pollen-wall proteins: physicochemical characterization and role in self-incompatibility in Cosmos bipinnatus. Proc. R. Soc. B 188, 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. J. Cell Set. 9, KNOX, R. B. & HESLOP-HARRISON, J. (1970a). Pollen-wall proteins: localization and enzymic activity. J. Cell Set. 6, KNOX, R. B. & HESLOP-HARRISON, J. (19706). Direct demonstration of the low permeability of the angiosperm meiotic tetrad using a fluorogenic ester. Z. Pfl. Ziicht. 62, KNOX, R. B., HESLOP-HARRISON, J. & HESLOP-HARRISON, Y. (1975). Pollen-wall proteins: localization and characterisation of gametophytic and sporophytic fractions. In The Biology of the Male Gamete (ed. J. G. Duckett & P. A. Racey), Biol. jf. Linn. Soc. 7, Suppl. 1, KNOX, R. B., HOWLETT, B. J., HESLOP-HARRISON, J. & HESLOP-HARRISON, Y. (1973). Pollenwall proteins: gametophytic and sporophytic fractions: their origin, localization and emission. Incompatibility Newsletter 3, LINSKENS, H. F. (1966). Die Anderung des Protein- und Enzym-Musters Wahrend der Pollen Meiose und Pollen Entwicklung. Planta 69, MATILE, P. (1968). Lysosomes of root tip cells in corn seedlings. Planta 79, PARISH, R. W. (1975). The lysosome concept in plants. II. Location of acid hydrolases in maize root tips. Planta 123, PEARSE, A. G. E. (1968, 1972). Histochemistry, Theoretical and Applied. 2 vols. London: Churchill. STOUT, A. B. (1931). Pollen tube behaviour in Brassica pekinensis with reference to self incompatibility in fertilization. Am. J. Bot. 18, THOMPSON, K. F. (1957). Self-incompatibility in marrow-stem kale, Brassica oleracea var. acephala. I. Demonstration of sporophytic system. J. Genet. 55, THOMPSON, K. F. & HOWARD, H. W. (1959). Self-incompatibility in marrow-stem kale, Brassica oleracea var. acephala. II. Methods for the recognition in inbred lines of plants homozygous for S alleles. J. Genet. 56, {Received 2 December 1975)