Pollen—Pistil Recognition: New Concepts from Electron Microscopy and Cytochemistry

INTERNATIONAL REVIEW OF CYTOLOGY. VOL. YO

Pollen-Pistil Recognition: New Concepts from Electron Microscopy and Cytochemistry C. DUMAS,*R. B. K N O X , ~AND ' T. GAUDE* *Dt!partement de Biologie VegPtale et C . M . E . A . B . G . ,Universite' Claude BernardLyon I , Villeurbanne, FrrInre, and 7"Schoolof Botany, University of Melbourne, Parkville, Victoria, Australia Introduction , , . _ .. _ . ,. _ .. ............ The Mature Viable Pollen G ............ 111. The Receptive Pistil. . . . . . . ............ ............ IV. Male-Female Interactions, . A. Attachment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Hydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The Progamic Phase of Fertilization.. . . . . . . . . . . . . . . . . . . . . . D. Pistil Interactions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. The Callose Rejection Response. . . . . . . . .............. F. Molecular Basis for Pollen Infomlation and Pistil Read-Out Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

11.

239 241 251 255 255 259 259 261 263 263 268 269

I. Introduction Following the simultaneous discovery of the mechanism of fertilization in flowering plants by the Russian S. Nawaschin and the Frenchman L. Guignard at the end of the nineteenth century, there has been considerable extension and clarification of the structures involved in reproduction. In terms of male-female recognition, i.e., interactions between pollen grain and pistil, perhaps two of the most exciting discoveries have been the demonstration that the pollen wall may be interpreted as a living structure containing enzymes and other proteins (Tsinger and Petrovskaya-Baranova, 1961), and, on the female side, the demonstration of the outer protein-containing layer of the stigma surface (Mattsson et al., 1974). Heslop-Harrison (1975) provided the first cellular model to explain male-female recognition in flowering plants. He used a new combination of data from electron microscopy and transposed concepts from animal cell-cell communication. This model, together with other more recent hypotheses, attempts to explain how the female partner is able to discriminate the right male partner to comply with the two principles of reproductive biology: increase in hetero239 Copyright 0 1984 by Academic Press. Inc. All right\ of reprcductiun in any form reserved. ISBN 0-12-364490-9

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C. DUMAS ET AL

zygosity and maintenance of stability of the species (see de Nettancourt, 1977; Frankel and Galun, 1977). When we examine these various models critically, we find there is very little evidence to support these usually complex hypotheses of pollen-pistil interactions (Dumas and Gaude, 1981; Knox, 1983a). In this review, we have attempted to clarify this important area of plant cell biology, and propose to interpret the interactions in terms of the key concepts. The first is that the proteins and other molecules carried by the pollen grain mainly in its wall provide prima facie information. The second is the read-out system possessed by the female partner to interpret this pollen information. Both systems operate under control of a complex genetic system, the S locus, which may be interpreted as a sexual recognition gene system (Fig. 1). As a consequence, if information and read-out are compatible, fertilization results. If not, depending on the extent, integrity, and efficiency of the read-out system, the interactions may be partly or totally blocked and incompatibility ensues. The pollen grain is thus unable to transfer its male germ unit, although it may be otherwise quite fertile. The incompatibility reaction that causes the blockage may be expressed as an active rejection response (Fig. 2).

I

INFORMATION

coA T

I

INTERFACE

READ-OUT SYSTEM

FIG. I . Pollen-stigma interface. During male-female recognition, signal molecules from the pollen coat transfer information to the stigma. These molecules interact with receptors on the stigma surface or within the style. An incompatibility between information and read-out system results in rejection characterized by inhibition of pollen germination or tube growth. Their compatibility provides for hydration which involves a flux regulated by osmotic differences between the both partners. This flow induces germination and tube growth. Analysis and precise localization of molecules constituting the information and the read-out system are necessary to modify the recognition system to obtain new plant types. ( I ) Flux of water from stigma to pollen grain. (2) Release of hydrophylic substances from pollen to stigma surface. Closed symbols, informational molecules of the pollen grain. Y-shaped symbols, molecules of the stigma read-out system.

24 1

POLLEN-PISTIL RECOGNITION CONTACT Pollen-Stigma Incongruity Barrier

-'

Pollen hydration

Pollen germination lnterspecific Barriers

Entry of pollen tube into pistil

I

lntraspeci fic Barriers

Growth of pollen tube to ovule

FIG.

2.

Barriers to fertilization: a simplified scheme.

The nature and control of pollen rejection suggest the operation of a sophisticated system to control self and nonself recognition in flowering plants. We have previously drawn parallels between the immune system and the major histocompatibility complex of vertebrates with the recognition systems of the most highly evolved plants, the angiosperms (Clarke and Knox, 1980; Dumas and Gaude, 1982). In this article, we will elucidate the nature and characteristics of pollen information and pistil read-out systems. The interactions between these two systems open a new and exciting area in plant cell biology, which will not only lead to a new understanding of reproductive biology but to the regulation and manipulation of fertilization and seed-setting for the benefit of mankind. 11. The Mature Viable Pollen Grain

The male partner is the mature viable pollen grain. It is virtually a dehydrated organism. The water content at time of dispersal varies widely among different pollen types, generally between 15 and 35% (Stanley and Linskens, 1974). The grains also have virtually no respiratory metabolism. Like spores or seeds, they

242

C. DUMAS ET AL.

FIG.3 . Bicellular pollen grain of Corylus uvellunu. Transmission electron microscopical (TEM) observation after PATAG treatment of the ultrathin sections showing the polysaccharidic nature of storage compounds. GC. Generative cell; VN, vegetative nucleus. X5700. (Photograph from M. H. Simard, Lyon I . )

utilize this means for dispersion and species survival (Frankel and Galun, 1977). Each grain is surrounded by a complex wall, which not only has mechanical protective properties, but has coat materials (pigments) which screen the hereditary material from damaging UV light (see Zandonella et al., 1981). About twothirds of pollen types are bicellular (Brewbaker, 1957), carrying the progenitor cell of the male gametes (Fig. 3). The remainder are tricellular, and the pair of sperm cells are borne directly within the vegetative cell at maturity e.g., in oil seed rape, Brussica (Fig. 4).

POLLEN-PISTIL RECOGNITION

243

The significance of this difference in the structure of the hereditary materials of the pollen grain becomes apparent when the pollen germinates. In tricellular types, the metabolism of the grain is geared to take advantage of its lead in development, and pollen tube growth rate may be very rapid, with a single phase, so that fertilization may occur within minutes of pollen deposition on the stigmas (20 minutes in grass pollen, 45 minutes in sunflower) (Heslop-Harrison, 1979a,b; Vithanage and Knox, 1977). In contrast, in bicellular types, metabolic changes are needed during germination (Mascarenhas, 1975) as well as cell division. Tube growth may be biphasic (Mulcahy and Mulcahy, 1983). Initially growth is slow, becoming more rapid after about 7 hours in Petunia. CsnSequently, fertilization requires at least 24 hours after pollination. For successful fertilization, pollen must be viable. Even if we collect pollen from a freshly opened flower, how can we be sure it is viable? Viability has traditionally been assessed by success in fertilization and seed set, but this is a time-consuming and laborious process. Today there are more rapid methods. I n vitro culture methods involve germination of pollen on an agar medium (semisolid) or in liquid culture (see Knox, 1983a,b). The medium comprises a carbon source, e.g., sucrose and essential mineral oligoelements including traces of boron and higher amounts of calcium. Within a time period of about 24 hours, it is possible to assess such parameters as percentage germination, pollen tube length, and growth rate. Unfortunately this test is applicable mainly to bicellular pollen types (e.g., Williams et d., 1982), as most tricellular pollen types show a very low percentage germination in vitro (Bar-Shalom and Mattsson, 1977; Roberts er u / . , 1983). Staining tests are also applicable to pollen, but these detect the presence or absence o f cytoplasm, so are indicators of pollen sterility rather than viability. The most rapid test available is the fluorochromatic reaction (FCR) first employed for pollen by HeslopHarrison and Heslop-Harrison ( 1970). The method requires fluorescence niicroscopy which limits its applicability in the field. The selective permeability properties of the plasma membrane of the pollen grain is tested with respect to fluorescein diacetate, and the presence of intracellular esterase activity. A correlation has been demonstrated between FCR data and pollen germination in vitro (Shivanna and Heslop-Harrison, 1981). These tests of pollen viability all involve loss of the sample tested, which is no longer available for subsequent breeding tests or other experiments. A new method developed by Dumas et LII. (1983) overcomes this problem. It depends on the use of nuclear magnetic resonance spectroscopy (NMR). It is based on the changes in water content of the pollen as it becomes nonviable. Traditional methods for estimating water content are unreliable. In nonphotosynthetic tissue, such as pollen grains, expression of water content on a dry weight basis is usually satisfactory, but is unsatisfactory for the same reason if expressed on a fresh weight basis. The most accurate method to determine cellular water content

245

POLLEN-PISTIL RECOGNITION

today is provided by thermigravimetric analysis with isotherms at 85 to 90°C until there is no further mass change. These estimates are accompanied by pollen viability tests by FCR. With this method, values for pollen water content as low as 5.6% have been obtained for pollen of Poputus nigru, and 16% for Brussicu oteraceu. Dead pollen apparently acts like a sponge in absorbing atmospheric water (Dumas er ul., 1984a). The NMR is employed to test the evolution in water content that occurs during loss of viability, e.g., in Brussica pollen (Dumas er al., 1983). The method provides data that are rich in structural as well as dynamic information. It utilizes the principle of resonance of the hydrogen atomic nucleus, [ H , when placed in an intense magnetic field. In the liquid state, the NMR signal has a narrow linewidth (approx. 1 Hz) while in the solid state (as with a dry pollen sample) a broad line-width (approx. 600 Hz) may be obtained. A negative correlation has been obtained between the values of the spin-spin relaxation time ( T I ) and the percentage viability in Brassicu pollen (Dumas ef u l . , 1984a). Relaxation times are computed from the formula 1 / T , = P ,[ I / T 2 ( h ) ]+ ( 1 - P I)[ 1 /T2cf)]where b is bound water molecules, f' is free water molecules, P I is proton spin lattice relaxation time, and T2 is proton spin-spin relaxation time. A model has been employed which includes two types of cellular water, bound water (low T2 values) and free water (high T , ) values. The expectation i s that living pollen contains more bound water than dead grains. Loss of pollen water content has been found to occur in threc steps (Figs. 5 and 6), following increasing temperature treatment and aging at room temperature. NMR results are closely paralleled by loss o f viability data from FCR tests of the same pollen. Thus, loss of viability is correlated with water content. Also. there is no water content expenditure in viable pollen under isothermic conditions, suggesting that water content is regulated within certain limiting temperatures commonly experienced by the pollen. This characteristic is perhaps mediated by the plasma membrane or the wall of the pollen grain with its viscous pollen coat. This may be an inlportant adaptation tor pollen survival during dispersal. A fraction of liquid water (vital water) may be eliminated through the porous plasma membrane of dead grains (Burke c't N I . . I976), but in a living grain the plasma membrane is an effective barrier. What i h the state of the plasma membrane o f mature pollen'? Two types are known: ( I ) those with a poro~isand ineffective plasma membrane, associated generally with a low water content and short viable life. e.g.. Secrrlr pollen (Heslop-Harrison. I979a,b): and ( 7 ) those with a continuous and effective plasma membrane, associated generally with a high water content and longer viable life. e . g . . Brussicw pollen (Dumas and Gaude, 1983). Here, a typical particulate structure characteristic of biological ~~~~

Fic;. 4. Tricrllular pollen grain of.Brmsictr olrrciwtr. TEM cihwwtion after hcavy metal u l t contrast. SCI and SC2. sperm cell\. VN. vrgriativc nucleu\. X S 9 0 0 . IFroni Dumaa CI o l . . 1984h.)

246

C. DUMAS E T AL.

I

0

\*

1

0

,

I

C

'

'

"

5

I

I

I

"

10

'

"

"

+

15

Days

FIG. 5 . Loss of water content during the pollen aging in Brussicu. Three steps (A to C) are visible. au. Arbitrary units. (From Dumas c( u / . , 1983.)

membranes has been detected in the plasma membrane by freeze-fracture (see Fig. 7 and Dumas et al., 1984a). The first type belongs to plants whose pollen is dispersed in air currents; the grain wall is generally thin with pollencoat reduced or absent (e.g., poplar, Populus, Fig. 8a). In the second type, the pollen is generally dispersed by specific animal vectors, and the wall is thick and reinforced, with a copious covering of sticky pollen coat (e.g., Brassicu, Fig. 8b).

t

100

50

Days

F K . 6. The viability of Bnrs.tic.rr pollen grain decreases from 90 to 0% after 2 week storage in a small closed elas\ container at room temperature (20-23°C). Here also three steps ( A to C ) are visible. (From Duma5 c/ d . . 1983.)

POLLEN-PISTIL RECOGNITION

247

Differences in the chemistry of the pollen coat are suggested by the ultrastructural studies of Hesse (1979. 19801, who has detected “active” and “inactive” types distinguished by their electron density and heterogeneity, and site at the exine surface. While the pollen envelopes play a critical role in regulating water loss and hence grain viability, they are also the site of initial contact with the stigma surface in male-female recognition. What potential has the wall in recognition’? It contains extracellular enzymes-as shown by Tsinger and Petrovskaya-Baranova (1961) and confirmed and greatly extended by Knox and Heslop-Harrison (1969, 1970). Later, further work showed that the wall proteins occurred in two quite different domains (Heslop-Harrison cr a/. 1973, Heslop-Harrison, 1975; Knox et a / ., 1975): ( I ) diploid parentally specified proteins held in a labyrinth of arcades in the outer wall, the exine (sporophytic domain); and (2) haploidspecified proteins laid down in the inner wall layer, the intine (gametophytic domain). A considerable number of proteins and glycoproteins (proteins with sugar chains attached covalently) are present in the pollen wall (see Table I). The proteins housed in the exine are first to make contact with the pistil, while those in the intine may be released more gradually during germination (see Table 11).

FIG. 7. Freeze-fracture of Brassic-u pollen wall showing the plasma membrane of the vegetative cell with a typical particulate structure characteristic of biological membranes. 1. Intine: pl, plasmalemma; arrowhead. direction of the shadowing. X83.700. (From Gaude. 1982.)

248

C. DUMAS ET AL.

FIG. 8. TEM observations of the pollen wall from the two types of grain according to their water content. (a) Pollen wall of Pcpirlus alba possessing a low water content. The exine is thin with a reduced pollen coat. The cytochcniicsl treatment used, e.g., cationized ferritin, allows visualization of the presence of numerous polyanionic sites on the exine surface. X25.000. (Photograph from M. Gaget, Lyon 1 .) (b) Pollen wall of Brassica oleracea grain with high water content. The thick exine carries a copious and sticky pollen coat which may induce adhesion between grains. E, Exine; I , intine; PC, pollen coat. X 12,300.

TABLE I NATUREOF SOME PROTEINS LOCAL.17.ED I N [ N T l N t POLLENGRAINS"

AND

EXINt: SITES

Ol-

Present in Protein Dehydrogenases NADH and succinic dehydrogenase Oxidases Cytochrome oxidase Transferases Phosphorylase Ribonuclease Hvdrolases Acid phosphatase Amylase Cellulase (p-I .4-glucanase) Esterase Invertase (p-fructofuranosidase) Polygalacturonase (Pectinase) Protease Allergens Ragweed Antigen E

Intine

Exine

-

+

-

+

(Rapidly diffusible)

+

-

+ +

-

+

(Rapidly diffusible)

+

+

(External to plasmalemma) (Rapidly diffusible)

+

+

+

+

<'From Knox el al. (1975). TABLE II AVERAGE TIMEOF EARLIEST DETtCTABLE PROTEiN Dlr;kLlSlON

Species and pollen type Alopecurus prarensis. Dacfvlis glo,nerma. Gramineae (tectate; single operculate aperture; smooth exine) Silene tdgaris. Caryophyllaceae (tectate; many operculate apertures; moderately smooth exine) Cosmos bipinnaius. Compositae (tectate; three colpi: spiny exine) Ambrosia irifida, Compositae (tectate; three colpi; exine with low spines) lbcris sempervireris, Cruciferae (murate; three colpi) Hibiscus rosa-sineiisis. Malvaceae (tectate; many nonoperculate apertures; moderately smooth exine)

UFrom Heslop-Harrison el al. (1975b).

FROM

POLLEN WALL."

Emission of sporophytic fraction from sexine sites (seconds)

Emission of gametophytic fraction from intine sites (minutes)

<25

2-3

<30

1-5

2-5

10-20

2-5

-0.5

<20

6- 10

<30

4-5

250

C. DUMAS ET AL.

There is thus evidence for a considerable number of potential informational molecules at the surface of the male partner. There is now biochemical and immunological evidence that many are specifically synthesized in the pollen grain, while others are common with those of somatic tissues of the same plant, e.g., Gladiolus (Clarke et al., 1977), Prunus (Raff et al., 1979), and Zea (Porter, 1981). Is there any evidence for a recognition system which might transmit signals from the pollen surface to the plasma membrane or sperm cells, or for structures at the surface of the pollen grain which may be involved in signaling with the pistil? Recently Gaude ( I 982) detected a surface exinic layer which may fulfil at least some of these properties. It has some of the characteristics of a membrane, including trilamellar structure, and characteristic appearance by freeze fracture microscopy, and coats the surface of the exine in mature pollen. This layer could have the potential to provide direct membrane-membrane contact with the stigma surface (Gaude and Dumas, 1984). Ultimately receipt of signals or their emission must reside with the nucleus of the vegetative cell or the sperm cells themselves. Two laboratories have now demonstrated the presence of physical connections between these elements of heredity. In Plumbago zeylanica, one sperm cell is linked by a long connection to the embayments of the nucleus of the vegetative cell (Russell and Cass, 1981). The pair of sperm cells are held within a common but discontinuous wall. We have recently demonstrated a similar nuclear association in Brassica oleracea pollen, but here the connections are multiple (Dumas et al., 1984b). Long processes, pseudopodia, link the sperm cells together in a tail-tail configuration, rather than in a head-tail or head-head association. The pseudopodia of the sperm cell adjacent to the vegetative nucleus are apparently contiguous with the nuclear envelope (Fig. 9). We have termed this complex the male germ unit, as it appears to be preprogrammed for effective fertilization.

FIG. 9. Scheme of the male germ unit as described in Brassica pollen. ps, Pseudopodia; SCI and SC2, sperm cells; VN, vegetative nucleus. (Built from Dumas et al., 1984b.)

POLLEN-PISTIL RECOGNITION

25 1

A further refinement with important consequences for mode of transmission of heritable organelles, e.g., maternal inheritance, is that the two sperm cells within one grain may exhibit preferential transmission of plastids and/or mitochondria. In Plumbago, mitochondria are largely restricted to the sperm cell adjacent to the vegetative nucleus, while the second sperm cell contains most of the plastids (Russell and Cass, 1983). The significance of this arrangement depends on which of the pair of sperm cells fuses with the egg, and whether any sperm cytoplasm is transmitted. In Brassicu olerucea pollen, there are no plastids in the sperm cells, and mitochondria are largely confined to the sperm adjacent to the vegetative nucleus (Dumas et a / . , 1984b). Thus the maternal inheritance of plastids observed in several genera of Cruciferae may be explained by their absence in the sperm cells. Hagemann (1979) has classified the generative cells of some angiosperms into three types: ( I ) those that completely lack plastids, e.g., Antirrhinum, Gossypium, Lycopersicon, Mirabilis, and Zea; ( 2 ) those that contain no plastids at maturity since they degenerated during differentiation, e.g., Beta, Hostu, Mimulus, Oryza, and Solanum; and (3) those that contain numerous plastids, e.g., Oenothera, Pelargonium. The patterns of maternal transmission of plastids by sperm cells have been reviewed by Sears (1980), who found four types: ( 1 ) exclusion of plastids during spermatogenesis, (2) loss from motile sperm, (3) exclusion during fertilization by sperm cleansing, and (4) degradation within the embryo. All these conclusions have been reached from the study of relatively few genera of angiosperms. They show a wide variety of different patterns of behavior, suggesting that cytoplasmic transmission by the sperm cells, as with nuclear, is far from a chance event. In Brusssica sperm cells, modifications have been observed prior to maturation within the anther that may be analogous to capacitation in mammalian sperm cells. Dramatic changes occur at the membrane interface between sperm and vegetative cell, which we have interpreted as the possible acquisition of recognition capacity, in a manner similar to the glycocalyx of animal cells. Indeed, the sperm cells of Brassica are remarkably similar to animal sperm cells, since they have no cell wall and no plastids (Dumas et a/., 1984b). The male germ unit thus shows specializaticn at both the nuclear and cytoplasmic levels, suggesting that future work may reveal membrane specificity and the means for cellular communication. 111. The Receptive Pistil

The female partner is the receptive pistil. This structure not only houses the female gametes, but also receives the pollen, and allows for germination and pollen tube growth and selective discrimination of right from wrong pollen types for successful fertilization and seed set. There are three important parts: (1) the

252

C. DUMAS ET AL.

stigma, where pollen is received, and whose surface may possess receptors capable of identifying the pollen type; (2) the ovary, a special female germ unit, is contained within the parental tissue of the ovary where the syngamic stage occurs in the ovule(s). After fertilization, the germ unit provides the seed, while the ovary provides the fruit; and (3) the style, which provides a mechanical facilitation pathway for the pollen tube during its traverse from the stigma to the ovary and during which the main part of the progamic phase occurs. The cytology of these structures has recently been extensively reviewed (Tilton and Horner, 1980; Knox, 1983b; Dumas and Gaude, 1983); here, we will consider only the aspects important for intercellular communication. The stigma is a gland, covered by specialized receptive cells, usually elongate papillae (see reviews by Heslop-Harrison and Shivanna, 1977; Heslop-Harrison, 1981). There are two broad, but overlapping types: wet stigmas which bear a copious secretion of exudate and dry types covered by a thin film of adhesive material termed the pellicle (Mattsson et a f . , 1974). In wet stigmas, both cytological and biochemical analyses have been carried out, and two subtypes identified. In the first, e.g., Lilium, the exudate is hydrophilic. Nearly 99% of the exudate is polysaccharides, proteins, and water (Aspinall and Rosell, 1978; Gleeson and Clarke, 1980a,b). Among the polysaccharides, more than 90% of the residues are galactose, arabinose, rhamnose, and glucuronic acid. One of the components is a stickly and much branched polymer, containing 0-rhamnopyranosyl-( 1 + 4)-glucopyranosyl uronic acid-( 1 + 6)galactopyranose (Aspinall and Rosell, 1978). This arabinogalactan has a galactose:arabinose ratio of 2: 1 (Gleeson and Clarke, 1980a,b). The cytological route of secretion of the exudate involves a granulocrine pathway of secretion, from ER and Golgi apparatus through the plasma membrane (see Dashek et al., 1971). This is a classical route for glycoprotein secretion (Chrispeels, 1976). A similar mechanism has been found in Aptenia stigmas (Kristen, 1977). In the second type of wet stigma, the lipophilic type, the most detailed analyses have been carried out in Forsythia. The exudate includes neutral lipids, especially triglycerides and fatty acids C, to C,,, terpenes, and phospholipids (Dumas, 1977). This exudate is sticky, acting as a liquid cuticle (as in Petunia, Konar and Linskens, 1966a,b). This prevents stigma dehydration in the absence of a cuticle. The route of secretion is mainly holocrine in type, involving smooth ER in the biosynthesis step, vacuoles in the transitory vacuolation period, and periplasmic accumulation before transport through the cell walls. Among dry-type stigmas, only a small number of types have been investigated by transmission electron microscopy (see Heslop-Harrison, 1981). A classic example is the stigma of Brassica, whose pellicle layer was first to be discovered by Mattsson et ul. (1974) and its properties further characterized by HeslopHarrison et al. (1975a,b). The cytoplasm of the stigma papilla is surrounded by a plasma membrane, a thick polysaccharide wall, a discontinuous cuticle, and the

253

POLLEN-PISTIL RECOGNITION

outermost layer, the pellicle. The pellicle is heterogeneous in ultrastructural appearance. In Brassica stigmas, it is not visible by transmission electron microscopy with Reynold's stain. Specific cytochemical methods need to be employed to visualize the pellicle (see Table 111). In other genera, this is not the case: in Populus and Saponaria the pellicle is readily detected by Reynold's stain. An interesting feature of the techniques employed to reveal the pellicle in Brassica is that not all give uniform continuous binding. Both Con A and mannosyl-ferritin bind in distinct patches, perhaps indicative of a capping-like phenomenon that occurs on animal cell surfaces. This may indicate that the pellicle is capable of changing its state. TABLE 111 ELECTRONMICROSCOPE CYTWHEMICAI.TREATMENTS USED TO V I S U A L ~THE Z ~ Pti.i.ici.t. DRYSTIGMA Srecits Treatment Localization of enzymatic activities Nonspecific esterasel'

ATPase Ltxalization of a pemieahility barrier Colloidal lanthsnum nitrate

Species

IN

Reference

Mattsaon ct ul. ( 1974)

Heslop-Harrison ( I97Sa) Caude (1982)

cf

01.

Heslop-Harrison

CI

id.

(197Sa) Gaude (1982)

Localization of polyanionic SltCS

Cetylpyridiniuni chloride Cationized ferritin Localization of glyciwmjupatca (u-iiiannosyl and aplucosyl rcsiducs) Con A-peroxidase DAB

Con A-iron dextran Con A-ferritin Con A-niannosyl ferritin

Clarke 1'1 trl. (19x0) Caudc (1982)

Hcrlop-Harrison (1976) Pettitt (1980)

Herd and Beadle (1980) Clarke and Knox ( 1978) Clarke cv ul ( 1 980) Caude (19x2)

"The presence of a nonspecific estcrase activity on the surface of dry stigmas has been extended for all species (Heslop-Harriscin and Shiviinna. 1977; Heslop-Harrison. 198 I ).

254

C. DUMAS ET AL.

An interesting feature of Con A binding to the pellicle is that it has been demonstrated to be specific only once (Knox et ul., 1976), i.e., capable of inhibition by specific sugars. This experiment was carried out by biochemical analysis; all subsequent cytochemical analyses by transmission electron microscopy have given a similar density of reaction product in test and controls. Exceptions are fluorescent-labeled lectin in Gladiolus (Knox et al., 1976) and peroxidase-labeled lectin in Phalaris (Heslop-Harrison, 1976). In the other cases (Table III), the methods may not be sensitive enough to detect any specific binding above background adhesiveness of the stigma surface. Ontogenesis of the stigma pellicle remains to be convincingly demonstrated at transmission electron microscope level. Some properties of the pellicle during its transition to receptivity were demonstrated by Heslop-Harrison et al. ( 1975a). An important feature of stigma function is the effective period of pollination, i.e., when the stigma is receptive to the right kind of pollen for seed setting. The duration of this period varies widely: from a few hours in an Australian mimosa, Acaciu retinodes (Bernhardt et al., 1983), to nearly 10 days in the grape vine, Vitis vinifera (Carraro et al., 1979). The age of the flower, the time of day, and the presence or absence of stigmatic exudate all may influence receptivity. How is stigma receptivity determined experimentally? There are three basic ways: ( 1 ) the morphological appearance of the stigma, including papillar movements, e.g., Hibiscus (Buttrose et al., 1977); (2) correlation with seed-setting ability; and (3) differential staining in dry-type sitgmas (see Table 111). In some cases, the end of receptivity may be indicated by the appearance of the cell wall polysaccharide callose which spreads progressively across the stigma, e.g., avocado (Sedgley, 1979). This may be interpreted as indicating the aging or senescence of the stigma cells. The ovules, of which there may be one or many within the ovary, also have a distinct period of viability, when they are prepared to receive pollen tubes. leading to successful fertilization. In pome and stone fruits, including avocado, the loss of viability of ovules for pollen tubes is indicated by the cytological appearance of callose in the cell walls, and its progressive spread across the cells of unpollinated aging ovules (see Dumas and Knox, 1983). The callose may again be an indicator of tissue senescence, perhaps through control of cellular autolysis by hydrolytic enzymes. An important feature of ovule viability is that it is part of the pistil viability calendar. It is possible for the growth period of pollen tubes in apple pistils to be so delayed that the ovules have lost viability by the time the pollen tubes reach them (Anvari and Stosser, 1978, 1981; MartinezTellez and Crossa-Reynaud, 1982). For pollination to lead to successful fertilization, the stigma must be receptive and the ovules viable. In most reproductive systems, the ovules contain fully differentiated female gametophytes-the embryo sac. These are generally present when the stigma is receptive and pollination anticipated. However, in a few groups of angiosperms,

POLLEN-PISTIL RECOGNITION

255

and in most gymnosperms, the ovary does not commence differentiation until after pollination, when pollen tube growth in the style has commenced. The pollen tube enters a period of dormancy at the base of the style until ovule differentiation is complete. This system occurs in hazelnut, oak, and certain types of orchid (see Knox, 1983a,b). In understanding the structure of the female germ unit, perhaps the major advance has come from recent studies of the embryo sac by transmission electron microscopy. Jensen (1974), a pioneer in this field, developed the concept of the female gametic complex, incorporating the egg cell with its pair of synergids. These cells are the first to be contacted by the male germ unit, after release from the pollen tube. The central cell needs to be added as part of the female germ unit, because of its role in endosperm formation following double fertilization. The central cell is surrounded by bounding membranes, while the synergids and egg cell have apical regions where the cell wall is absent, permitting direct membrane contact with the sperm cells.

IV. Male-Female Interactions What are the cellular events that occur in the pollinated pistil during the progamic phase leading to fertilization'? First of all, pollen is deposited on the stigma, where its species- and genotype-specific information is read-out by the stigma or other pistil tissues. This information and read-out system is encoded in a complex genetic system, of which the most common and simplest type is the monofactorial S locus, with many alleles (see de Nettancourt, 1977; Lewis, 1979). It is likely that the S supergene controls many processes of reproduction, including certain interspecific incompatibility responses, as well as preventing self-fertilization. In any event, pollen acceptance or rejection is based on the consequences of a dialogue between the gene products controlled by the S supergene. The process as a whole is a system to achieve fertilization and options for continuing or aborting the interaction occur at each subsystem encountered (see Knox and Clarke, 1980; Williams, et al., 1982). A. ATTACHMENT Adhesiveness is a fundamental cellular property which plays an important role in cell-cell interactions and recognition (Frazier and Glaser, 1979). Pollen adhesion to the stigma depends on the deposition and sedimentation of the pollen with the substratum (biological or physical) and on the subsequent formation of attachement bonds. It is accepted today that pollen adhesion is achieved by a series of steps, namely pollen deposition, contact, attachment, and hydration. It is

256

C. DUMAS ET AL TABLE IV PHYSICOCHEMICAL APPROACH TO POLLEN-STIGMA ADHESION SUGGESTED BY COMPOSITION Ol- A N IDEAL ADHESIVE ACCORDING TO R ~ Y N O L D ( 1 S971 )"

T H ~

Candidate

Component

Characteristic

Adhesive base Branched polymer of high molecular weight Plasticizer Thickener

Tackifier

Detergent

Pollen surface

Stigma surface

'?

Arabinogalactans

Prevent adhesive becoming brittle Increase viscosity of adhcsive

Diffusible mono- Free nionosacsaccharides charides Glycoproteins Glycoproteins

Resin-like cornpounds enhancing adhesive properties Wetting agent

Pigments of the pollen coat

Glycolipids

?

Glycolipids

Reference Clarke et a / . (1979); Gleeson and Clarke (1980a,b) Clarke et a / . (1979)

Knox et a / . ( 1976); Gaude (1982); Nishio and Hinata (1978) Heslop-Harrison (1968); Clarke et al. (1979) Clarke et a / . (1979); Gaude (1 982)

<'Based on Clarke et a / . (1979) and Dumas and Gaude (1982)

likely that each of these steps gives rise to an increasing degree of adhesion or binding (Dumas and Gaude, 1981). The contact step which initiates the interaction is probably mainly physical in nature, depending on surface charge phenomena. Five negative charges per 100 A2 are considered to be necessary for contact to be achieved (see Maroudas, 1977). Electrostatic forces have yet to be demonstrated on pollen surfaces, although histochemical data may be interpreted as indicating the presence of negatively charged groups within the exine (see Knox, 1983a,b). Some of the parameters have also been reviewed by Wottiez and Willemse (1979). Attachment takes two forms: nonspecific (irreversible) and specific (reversible). Nonspecific attachment is considered to be mainly chemical in nature. Clarke et al. (1979) pointed out that all the components of an ideal adhesive are present when both pollen and stigma surfaces are taken together (see Table IV). Specific attachment has often been interpreted from work with animal cells as a reversible attachment, e.g., with lectins or glycosyltransferases. Evidence for specific adhesion has come from binding of lectins to Gladiolus stigmas (Knox et al., 1976) where binding was assessed using radiolabeled lectin, and from in-

257

POLLEN-PISTIL RECOGNITION

terpretation of pollen adhesion assays in Brassica (Roberts et al., 1979; Stead et al., 1980). The pollen-stigma interface is very different in terms of adhesion between compatible and self-incompatible situations. The size of the interface is much reduced following self-matings, but greatly increased following cross (compatible) matings. One interpretation of these data is that the physical forces governing adhesion are strongest in compatible matings where there is specific adhesion. What kind of factors may be involved in specific adhesion? Several classical molecular models have been proposed to explain the nature of the bonds between cell surface components in specific or nonspecific adhesion. Three models are especially important. The first is the glycosyltransferase model of Roth (1973). He suggested that the enzymes present on one cell may interact with appropriate substrates on a second cell to form a stable enzyme-substrate complex, which would constitute the bond of attachment. While no glycosyltransferase activity has yet been found in pollen or stigma surfaces, an enzyme with associated glycosyltransferase activity has recenty been located in the pollen wall of Brassica, namely P-galactosidase (Singh et al., 1983). Further, pollen grains defective in the enzyme are extremely inefficient in adhesion to the stigma surface. The second model was suggested by Bowles (l979), and deals with the participation of lectins in intercellular communication. Lectins are proteins which exhibit specific, reversible carbohydrate-binding activity and can be multivalent (Goldstein el al., 1980). In this model, the lectins are membrane-bound, but their receptors may be similar or soluble components. Two main states can occur: either the complementary pairs are closed and self-neutralized, or alternatively the pairs are open and not complexed. This model is not incompatible with the first, since regulation may be mediated by glycosyltransferases, glycosidases, or proteases. The question arises whether lectins are present at either pollen or stigma interface'? Both lectins and lectin-like compounds are present (Table V) while

LECTINSOR LECTIN-LIKt

FOUND

Localization

TABLE V (AGGLUTININS A N D MITOGENS) IN POLLEN A N D PISTIL

COMPOUNOS

Activity

Reference

Pollen Cyiiodon dactylon

Platanus acrrifolia Brassica oleraceu Populus curamericana Pistil Primula obconica

Leukocyte agglutinins Lymphocyte mitogens Hemagglutinins Hemagglutinins

Lindberg el a/. (1982) Anfosso er a / . ( I 983) Gaude et a/. (1983) Gaude et a / . (1983)

Hemagglutinins

Golynskaya et ul. ( I 976)

25 8

C. DUMAS ET AL

CON A BINDING

ON

POLLEN

TABLE VI STIGMA SHOWING THE PRESENCE OF GLYCOSYL RESIDUES BOTHINTERACTING SURFACES

AND

Localization Pollen Grasses Brassica oleracea; B . napus Saponaria officinalis

Stigmao Gladiolus gandavensis

ON

Treatment

Reference

Double diffusion tests; immunocytology FITC-Con A; Con A-mannosyl ferritin FITC-Con A

Watson ef a / . (1974)

Helianthus annuus

FITC-Con A FITC-Tridacnin FITC-Con A

Brassica oleracea; B . napus Saponaria afficinalis

FITC-Con A FITC-Con A

Gaude ( I 982) Gaude and Dumas (unpublished data)

Knox et a/. (1976) Clarke ei al. (1979) Vithanage and Knox ( 1977) Gaude (1982) Gaude and Dumas (unpublished data)

“Electron microscope cytochemical treatments using labeled Con A are listed in Table 111.

polysaccharides and glycoconjugates with sugar residues that may bind to labelled lectins (Table VI) are also present. Unfortunately, little is known of their chemical nature and their interactions at pollination. In vitru, lectins such as Con A may have widely different effects: stimulating pollen tube growth (Southworth, 1975), blocking pollen tube penetration of the stigma surface (Knox et al., 1976), and affecting the read-out of pollen information at the stigma surface (Kerhoas et al., 1983). The presence of agglutinins in Brassica pollen has been demonstrated by a pollen-rosetting technique with red blood cells (Fig. 10, and Gaude et al., 1983). This agglutination could not be inhibited by a range of mono- and disaccharides suggesting that the agglutinin may be membrane-bound, possibly in the plasma membrane or in the exinic outer layer. In contrast to this finding, Gaude et al. (1983) also found a soluble agglutinin in the diffusate of Pupulus pollen. Agglutination could be prevented by heating extracts to 80°C or by trypsin treatment. Barondes (1980) devised the term cell adhesion molecules (CAMS) for those molecules capable of binding cells to other cells, or for extracellular binding components. CAMS may be nonspecific, binding all cells together regardless of origin, or highly specific to one kind of cell. Cell adhesion is considered to result from interactions between a surface protein and complementary receptors on other cells.

POLLEN-PISTIL RECOGNITION

259

FIG. 10. Scanning electron microscopic observation of the binding of native rabbit erythrocytes to a Brussicu pollen grain. Po, Pollen grain. X 1100. (From Gaude P I a/.. 1983.)

B . HYDRATION Hydration occurs in response to specific adhesion; water passes from the stigma to the pollen grain, and the subsequent enlargement of the grain constitutes the first morphological and physiological event of pollination. The water potential of a mature pollen grain is considerably lower than that of the surrounding moist substrate of the stigma surface. Consequently, water flows in the direction of decreasing water potential, from the stigma to the pollen. The grains swell, may undergo a change in shape, and become properly hydrated. The kinetics of the process in grass pollen have been elegantly demonstrated by Heslop-Harrison (1 979a,b).

C. THEPROCAMIC PHASEOF FERTILIZATION Several important biochemical modifications occur during the early events of pollen-pistil interactions. First, cutinases and probably carbohydrases are acti-

260

C. DUMAS ET AL

vated and lyse the stigma cell wall around the tip of the pollen tube. Active cutinases have been shown to be present in the pollen of nasturtium, Tropaeolum mujus (see Kolattukudy, 1981). The enzymes differ substantially from fungal cutinases, which also exhibit tip growth. In other systems, active cutinases were present only during pollen-stigma interactions, and could not be detected in the mature pollen (Heslop-Harrison, 1977). Perhaps the active enzyme is formed by subunits contributed from both pollen (pollen tube) and stigma. Christ ( 1959) provided a simple model to explain pollen-stigma interactions in Cruciferue in terms of cutinase activation. He considered that the cuticle acts as an important barrier in the self-incompatibility response of Cruciferae. He showed that two possible combinations were active: ( 1) in self-incompatible matings, active cutinase of pollen is inhibited by stigma, preventing cuticle erosion, and (2) in cross-compatible matings, an inactive precursor form in pollen is activated by stigma factors, leading to cuticle erosion. Later, Linskens and Heinen (1962) supported this hypothesis, noting that some erosion of the cuticle occurs in self-pollinations. The cutinase model alone is inadequate to explain the phenomena of pollen tube arrest following selfmatings (Dickinson and Lewis, 1973). After penetration, the pollen tube is guided through the intercellular system of the cell walls of the style, mostly comprising a fluid or mucilage. This extracellular system, whether it is in the form of a canal (e.g., lily), furrow (e.g., date palm), or solid tissue (e.g., tomato), serves as a mechanical facilitation pathway for the pollen tubes, often direct to the ovules themselves. Most pollen grains range in size from 20 to 200 km (an exception is certain seagrasses whose filamentous grains may be up to 5000 pm long (see Ducker er al., 1978). Yet they are capable of producing pollen tubes that may be several centimeters in length. How is this growth achieved'? The living cytoplasm, containing the male germ unit, is borne in the tip of the tube. As the tube grows, turgor pressure within it decreases, inducing physical stress which may result in the formation of a callose plug which seals the living tip from the remains of the pollen grain, and restores turgor pressure to the system. In this way, with continued growth, a series of callose plugs is formed, so that the elongating tube finally resembles a ladder. The question then arises whether the pollen grain has sufficient resources to sustain such growth? An elegant demonstration has been provided by Loewus and Labarca (1973). They used two different types of methodology: (1) in vifro pollen culture with incorporation of ['H]myoinositol (a pectin precursor forming the dominant wall polysaccharide in the tubes), and (2) in vivo tube growth in excised styles, where the tubes emerged into a medium containing the radiolabeled compound or it is injected into whole styles. The radioactive component accumulated in the style mucilage and became incorporated into the tube wall

POLLEN-PISTIL RECOGNITION

26 1

during its biosynthesis. (see also Miki-Hirosige and Nakamura, 198 1; Kroh and Knuiman, 1981). Because the pollen tube, as vector for the sperm cells, obtains part of its nutrition from the female tissues, it may be useful to compare it with a plant parasite. Why does the female tissue not reject this parasite? There is an obvious parallel in the acceptance of the fetus during pregnancy in mammals, controlled by the HLA system. The pollen tube may have a symbiotic association tolerated by the female tissue to ensure success of fertilization. In flowering plants, the pollen tube as vector of the male germ unit carries it to the female gametic complex, and employs the same strategy: an apparently passive transmission mediated by the pollen tube during its tip growth through the style. In contrast, less-evolved types of plants employ a more hazardous system of sperm cell transfer, which involves direct sperm-egg contact by swimming sperm cells in an aqueous medium. D. PISTILINTERACTIONS Information exchange and read-out for the stigma take place at the pellicle in dry-type stigmas. We have recently demonstrated (Kerhoas et al., 1983) the importance of the pellicle and its receptors in read-out of information from Brussica pollen. In this system, callose synthesis is triggered in the stigma cells within minutes of self (incompatible) pollination, but not by cross (compatible) pollination. The callose can be monitored by staining tissues with aniline blue fluorochrome, and fluorescence microscopy. It is thus possible to investigate the nature of the pollen information that induces the stigma response (see Dumas and Knox, 1983). Pollen information is provided by extracts of known protein content, obtained as whole pollen diffusates; after diffusing for a few minutes, the pollen is removed by filtration, and the extract applied as a drop to the stigmas. These diffusates contain wall-held proteins, glycoconjugates, and other molecules, i.e., pollen information. This is able to mimic the response of viable pollen in callose responses of stigmas. Self-pollen information induces a stigma callose response, while compatible information does not. When stigmas are first treated with the lectin, concanavalin A , known to bind to the pellicle in this and other systems, the subsequent response to self-pollen information is blocked. The lectin may mask the pellicle, so that the stigma is unable to read-out the pollen information. Also, when stigmas are first treated with a detergent, Triton X- 100, the subsequent response to self-pollen information is blocked. It is likely that the detergent modifies or partly solubilizes the pellicle, so that once again it is unable to read-out the pollen information. Does the pollen grain read-out stigma information'? Evidence from several systems where the pollen grain produces callose following self (incompatible)

262

C. DUMAS ET AL

but not after cross (compatible) pollinations (see Dumas and Knox, 1983) suggests that the pollen grain does receive information from the stigma, and responds to it. We must also assume, although there is as yet no conclusive evidence, that information exchange occurs at each diagnostic landmark in the pollination system (see Fig. 2 ) . There is some evidence that specific signals are exchanged between pollen or pollen tube, and a structure that is widely separated at the time of exchange: the ovule. Signals are received in the ovules of Petunia at about 9 hours and between 18 to 30 hours after pollination, in both cases, prior to pollen tube contact with the ovary (Deurenberg, 1976a,b). The signals are detected by increases in protein synthesis in the ovules of pollinated plants which do not occur in unpollinated control plants. The signals have the following characteristic features: ( 1) primary signal indicates arrival of pollen tube in style; and ( 2 ) secondary signal indicates whether self- or crosspollen is present in style. When concomitantly the ovule is viable, and the stigma is pollinated, one of the synergids within the gametic complex of the embryo sac is transformed. It changes dramatically in structure and composition, appearing to have degenerated. It is this synergid which the pollen tube finally enters. Jensen et al. (1983) have induced this change in vitro in one of the two synergids by treating cultured ovules of cotton with the plant hormone, gibberellic acid. They detected high levels of calcium in the transformed synergid, and draw a parallel with in vitro pollen tube growth experiments where calcium has been shown to have a chemotropic effect. In cotton, syngamy is made possible since the synergid and egg cell walls are incomplete around the apical parts of these cells. Sperm cell cleansing apparently occurs during syngamy in Petunia. Van Went (1970) has shown that the two sperm cells fuse with the plasma membranes of the egg or central cell, and only the sperm nuclei appear within the cells prior to nuclear fusion. During this germ unit confrontation, the question arises as to whether the pair of sperm cells fuse at random with egg or central cell, or whether there is some predetermined specificity. Russell and Cass (1983) claim this exists for fertilization in Plumbago. Certainly the structure and composition of the germ units in both Brassica and Plumbago suggest that the process is not random. Also, the mobility of the sperm cells within the pollen tube confers on them the advantage in selection, since the female partners are static as far as is known. However, how much reliance should we place on the possession of a particular heritable organelle as an indicator of sperm cell specificity? Does it in fact reflect any different binding specificity at the plasma membranes of the gametic complex? The demonstration of sperm cell specificity in structures that are only a few micrometers in size will require considerably more sophistication than has been employed to date.

POLLEN-PISTIL RECOGNITION

263

E. THECALLOSE REJECTIONRESPONSE Callose is implicated in the active rejection response observed in stigmas of self-pollinated Brassica, as noted earlier in this review. There have been considerable speculations on the possible role that the callose might play: ( I ) prevent tissue hydration through control of cell water equilibrium through antagonistic roles of calcium and potassium ions (Eschrich, 1975); (2) pool of nutrients available after hydrolysis in accordance with the transitory nature of callose deposits (Currier, 1957; Sedgley, 1977); or (3) active defense reactions in isolating or sealing pollen from the stigma (Aist, 1976; Lewis, 1980). It is possible that the compatible pollen grain might actively destroy the rejection mechanism through the action of a pool of enzymes following stigma surface recognition reactions (see Fig. 11). This means that callose will be actively and continuously degraded in a compatible pollination (Linskens, 1975, 1976). In the incompatible interaction, boron may be sequestered by callose, producing a boron deficiency leading to alteration of polyphenol metabolism. Phytoalexin-like components may then be synthesized (Lewis, 1980). The phytoalexin, rishitin, is known to inhibit pollen tube growth in vitro (Hodgkin and Lyon, 1979). The compatible situation has been compared with the host plant resistant to fungal disease (see Dumas and Knox, 1983). F. MOLECULAR BASISFOR POLLENINFORMATION A N D PISTIL READ-OUTSYSTEMS Most of the data available today concern the pistil read-out systems, and little is available for pollen information. We will review this latter material first. 1. Pollen Information Systems The existence of antigens with specificity directed toward the alleles of the S supergene of Oenothera orgunensis pollen was demonstrated by Lewis (1952). The S-specific antigen comprised about 20% of the pollen proteins and diffused from moistened pollen in isotonic media within 30 minutes (Makinen and Lewis, 1962). Individual pollen grains produced precipitates when sprinkled on gel containing S-specific antisera (Lewis et al., 1967). Unfortunately no further progress has been made with this system in identifying and characterizing the active protein. Linskens (1960) also identified antigens with S allele specificity in pollen of Petunia. A comparison of pollen antigens with those from other plant tissues has been made in Gladiolus (Clarke et al., 1977) and Prunus (Raff et al., 1979) which has a self-incompatibility system. Allergens, proteins with the ability to bind to a specific immunoglobulin E in man and provoke the allergic response (seasonal asthma and hay fever), have also been isolated from many types of pollen. Their function in the plant is unknown (see Howlett and Knox, 1983).

264

C. DUMAS ET AL

HOST/ PATHOGEN

0 CALLOSE

J.

ElHxH Callose degradation

Enzyme Pool

Callose accumulation

No inhibitor

Complexing

synthesis

of borate

Inhibitor synthesis (e.g., phytoalexin)

El COMPATIBLE

I

El INCOMPATIBLE

FIG. I 1 . Summary scheme showing current hypotheses concerning callose synthesis, degradation by hydrolytic enzymes, borate complexing, and inhibitor production (e.g., phytodlexin-like compound) in self-incompatible pollination. (See details in text; from Dumas and Knox, 1983.)

Enzymes present in the pollen wall may also be important components of the information system. In Brassica pollen defective for P-galactosidase, the efficiency of adhesion to the stigma is drastically reduced, suggesting that the enzyme plays a role in stigma communication (Singh et al., 1983).

POLLEN-PISTIL RECOGNITION

265

2. Stigma Read-Our Systems

In some cases, stigma read-out is located on the surface, at the pellicle. The read-out system appears to be S gene specified. Using isoelectric focusing methods, Roberts et al. (1979) demonstrated that the appearance of glycoproteins binding to the lectin Con A is correlated in time with the acquisition of selfincompatibility in Brassica. Previously, Nishio and Hinata ( 1977, 1979) had shown that a glycoprotein with S allele specificity (S, allele) showed a characteristic affinity for Con A. Later, Gaude (1982) found that Con A binds to the pellicle of Brassica, suggesting that the S-specific glycoprotein is pellicle located. Recently, Ferrari et al. (1981) isolated and partly characterized a glycoprotein specific for the S, allele. It has been found to regulate pollen germination in v i m , and to modify the behavior of compatible pollen at the stigma surface. Similar pollen growth inhibition may be obtained after treating the Brassica stigma surface with Con A (Kerhoas et af., 1983). Previously, S-specific antigens had been analyzed by immunodiffusion and immunoelectrophoresis (see Nasrallah, 1979) while the molecular size and nature of the S-specific macromolecules have been determined by a variety of physicochemical methods (Table Vll). It is likely that these potential recognition molecules may act in the stigma pellicle in Brassica. Their presence may be correlated with stigma receptivity, i.e., they provide an efficient, functioning read-out system. The Brassica system, controlled by a sporophytically inherited S gene system, with read-out and inhibition at the stigma surface, provides an ideal experimental system. In contrast, gametophytic systems, where only the site of rejection of pollen tubes is known, usually deep within the style, are much more difficult to approach experimentally. These systems appear to provide for retardation in expression of the read-out system. Is pollen information completely preprogrammed and available in the mature bicellular pollen grain? Most gametophytic systems are associated with this pollen type (Brewbaker, 1957). Furthermore, they generally have wet-type stigmas, in which the pellicle may be destroyed or partly solubilized, so that a new read-out system is needed. Where is it located? Is it adjacent to the site of tube inhibition'? Inhibition occurs within the mechanical facilitation pathway of the style-in furrow, canal, or transmitting tissue-in an intercellular mucilaginous medium. If the read-out system does not remain at the stigma, then this poses another problem. The pollen information system will have to be located at the growing tip of the pollen tube. It is also significant that tube arrest in gametophytic systems may occur at the point of transition into the parasitic state for the pollen tubes (Mulcahy and Mulcahy, 1983; Raff et a/., 1981).

266

C. DUMAS ET AL

TABLE VII NATUREOF MATERIAL WHICHCORRELATES W I T H S GENOTYPE DETECTED I N POLLEN OR STIGMA EXTRACTS<’ Nature of putative S gene product

System Sporophytic Brassica olrracea

Brassica campesrris

Gametophytic Oenothera organensis Petunia sp Prunus uviutn

Nicotiana aluru

Stigma: S2 antigen: glycoprotein; p/ high MW 54,000 Stigma: S antigen: Con A-binding glycoprotein; p/ 10.3; MW 57,000 Stigma: S22 antigen: Con A-binding glycoprotein; pl 1 I . I ; MW 60,000-65.000 Stigma: S7 antigen: Con A-binding glycoprotein: pl 10.6; MW 57,000 Stigma: S7 antigen: Con A-binding glycoprotein; pl 5.7; MW 57,000

Pollen: S allele corresponding antigens Pollen and style: S allele corresponding antigcns Style: S antigen: Lectin-binding glycoprotein; pl high; MW 37.000-39.000 Style: S allele corresponding products

Reference Ferrari et u / . (1981) Nishio and Hinata ( 1982) Nishio and Hinata (1982) Nishio and Hinata (1982) Nishio and Hinata ( 1979)

Makinen and Lewis ( 1962) Linskens (1960)

Mau et a / . (1982)

Bredemeijer and Blass (1981)

“Completed from Mau et a / . (1982)

In the pistil of Prunus uvium,which has a wet-type stigma, five antigens have been identified, including two in the style, antigens P and S (Raff et ul., 1981). Antigen P is common to all genotypes, while antigen S is specific to S,S, genotypes. Their properties are given in Table VII. Both are glycoproteins, and glycoprotein S strongly inhibits self-pollen tube growth in vitro (Williams et al., 1982). One question that arises is whether this component is an S gene product, or is it a receptor for S gene product from the pollen? The answer is not yet known, but several types of intercellular communication between the male information and the female read-out system may exist. Models have been proposed by Gleeson and Clarke (1980a,b). An elegant molecular model to explain S gene action was proposed by Lewis

POLLEN-PISTIL RECOGNITION

267

(1965): the oppositional or inhibitory model. The S gene is expressed in both pollen and pistil-ither producing identical dimers which fuse together to give an active tetrameric inhibitor; or complementary stereospecific molecules interact, the complex produced by fusion being inhibitory. Molecular interactions adapted for the control of hydration step of the pollen grain have been recently proposed (Gaude, 1982; Dumas and Gaude, 1983). Following the pollen-stigma contact, a reorganization of the pollen information and read-out system occurs (Gaude, 1982). This event may play a preponderant role in pollen adhesion and recognition processes in assuring control of the water flow from stigma to pollen. The pollen hydration level after pollination seems directly correlated to the acceptance or rejection of the grain, as shown by the work of Roberts et al. (1980). Once more, overcoming incompatibility by increasing the relative humidity confirms the importance of the hydration step in recognition mechanism (Carter and Mac Neilly, 1976). These facts led us to propose a molecular model to explain male-female recognition in sporophytic systems (Gaude, 1982; Dumas and Gaude 1983). This model is based on the control of the water flow from stigma to pollen by the reorganization of cell surface components of both interacting partners (Fig. 12). The pollen hydration

a

FIG. I ? .

Model of molecular interaction between pollen and stigma surface components in interface during compatible combination. The specific interaction between S-products from the pollen information and rcad-out system would lead to a reorganization o f cellular surface components. Lipids and proteins of the interface settle according to their respective affinities (electrostatic. hydrophobe. hydrogen liaiwna. van der Waals forces) The rnacrornolecular edifice thus formed would present facilitated ways for water flow and ensure a full pollcn hydration. The hydrated grain germinates and the pollen tube can fertilize the embryo sac. (b) Pollen-stigma interface during incompatible combination. The S-product interaction would induce the formation of a hydrophobic macromolecular structure which would constitute a barrier to efficient hydration of the pollcn grain. Such a weak hydrated pollen is rejected on the stigma surface. The pollen coat (PC) and the stigma pellicle (Pe) are composed principally by lipids (L). proteins. and glycoproteins ( P ) . Arrows indicate water flows from stigma to pollen.

Brc4ssic.u. (From Gaude. 1982.) ( a ) Pollcn-\tigma

268

C. DUMAS ET AL.

must no longer be considered as a simple physical phenomenon depending on an osmotic pressure difference between male and female organisms, but constitutes the first recognition event involving membrane-like structures (Gaude and Dumas, 1984). The interactions between these compounds and the modifications which are flowing would regulate the water flux and, thus, the acceptance or rejection of the pollen grain.

V. Conclusions In this new area of cell biology of fertilization in flowering plants, the concepts of pollen information interacting with pistil read-out systems are strongly supported by the available data. This is especially true for systems like Brassica where the major events of recognition occur on the stigma surface. Today, the data suggest that these systems operate through glycoconjugates and related molecules. This view is in agreement with recent work on animal systems (Koszinski and Kramer, 1981). These recognition molecules appear to act in a way analogous to antigen-antibody, lectin-sugar residue, or enzyme-substrate interactions. This analogy has been extended to include the possibility of direct membranemembrane contact between pollen and stigma surfaces in Brassica. Pollen information is generally regarded as being housed within the two domains of the pollen wall, i.e., in the pollen coat or within the intine polysaccharide matrix. In Brusuicu pollen, the membrane-like exinic outer layer described earlier in this review may contain many of the recognition determinants. On the female side, there is increasing evidence that the pellicle of dry-type stigmas, the remarkably plastic surface layer, appears to have many of the properties of a membrane, if not its structural characteristics. These two surface layers provide the possibility for direct contact and interactions, although these have yet to be demonstrated. These new concepts in plant cell biology are especially valuable in interpreting several techniques used empirically in plant breeding to overcome incompatibility responses. These methods involve modifications to the surfaces of pollen or pistil partners, either by physical or chemical treatments (see Clarke and Knox, 1978). Today, wc interpret these techniques as modifying the pollen information or pistil read-out systems (see Dumas and Knox, 1983; Dumas et d.,1984a). This approach is especially useful to begin to understand the physiology ofS gene action. Genetic manipulation ofthe natural route for fertilization and seed-setting becomes a practical possibility, through manipulation of the information or read-out systems. The possibilities of the utilization of the male germ unit for the transfer of genes through the vehicle of the pollen tube may be realized. We may anticipate rapid progress in the future in understanding and nianipula-

POLLEN-PISTIL RECOGNITION

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tion of fertilization through the characterization of mutants, such as that for pollen defective in the enzyme P-galactosidase (Singh er al., 1983). The use of mutants provides experimental systems in which the reproductive process is blocked at specific points, because of the expression of the mutant gene in the haploid pollen grains. The discovery that many isozymes of pollen are held in common with other parental tissues indicates the potential for gametophytic selection through the medium of the pollen tube at the haploid level (see Mulcahy, 1979). Thus the process of fertilization in flowering plants is open to manipulation for the benefit of mankind.

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