Friday, 18 October 2019

Pollen Pistil Interactions

Interactions

The structural features of the stigma and style have obvious implications in the biology of sexual reproduction. The major events that follow pollination are pollen recognition and the subsequent acceptance or rejection of the pollen grain by the stigma or of the pollen tube by the style. There are cellular mechanisms that operate to discriminate between the different types of pollen grains that dock on the stigmatic surface, germinate, and course through the style. These mechanisms ensure that only intraspecific pollinations are successful except when self-incompatibility genes act to prevent inbreeding. Intergeneric and interspecific crosses are frowned upon by the plant, which employs a different set of mechanisms to avoid such crosses. As shown by nearly all of the published work in recent years, the emphasis in developmental and molecular studies of pollen-stigma interactions has been on the postpollination behavior of the self-pollen grains and pollen tubes and on the isolation and characterization of genes involved in self-incompatibility reactions.

The concern of this section is with pollen-stigma interactions during compatible pollinations. Following compatible pollination, profound physiological changes occur in the pollen grain and in the stigmatic papilla and in signal transductions between these cell types in reciprocal acknowledgment of each other. The predominant changes reflect recognition between complementary molecules on the pollen grain and on the stigmatic papilla and the associated enzyme reactions. How these changes are translated into germination of the pollen grain will be explored now.

Pollen Adhesion

Interactions


Despite the fact that a variety of pollen grains are trapped on the stigmatic surface of a flower, the system favors the selection of only the compatible pollen. The changes that occur in the components of the pollen grain and of the stigmatic papilla after compatible pollination are now documented in a few cases and the general model of pollen recognition is known in outline. In this context, the adhesion of all kinds of pollen grains to the stigmatic papillae is clearly of considerable interest and presents a challenge to explain the basis of one of the most important manifestations of recognition.

Sufficient evidence has been adding up for some time now that one can assert that the idea of molecular recognition between pollen and stigma is not as unreasonable as was once thought, and several models are at hand to explain the phenomenon (Dumas and Gaude 1981). Lectins have been implicated as recognition molecules because of their wide distribution in the plant kingdom and their unique ability to bind saccharides and saccharide containing proteins in a highly specific way. The possibility that differences in the physical binding of the pollen to accessible glycoprotein sites in the stigma may regulate pollen adhesion is supported by the discovery that con-A binds to the pellicle of the stigma of Phalaris minor (Y. Heslop-Harrison 1976). 

The surface determinants of the stigma of Gladiolus Gandavensis contain a number of glycoprotein components and esterases, and the capacity to bind con-A is eliminated if these sites are occupied (Knox et al 1976; Clarke et al 1979). In Brassica oleracea, the initial contact of the pollen grain with the pellicle is through a superficial layer investing both the pollen and the exine coating (coating superficial layer). The pellicle appears to fuse with this layer as the first sign of adhesion (Elleman and Dickinson 1986). That some proteins responsible for pollen grain adhesion in B. Oleracea are held in the pellicle is supported by the observation that treatment of the stigma with protease adversely affects the adhesion of pollen grains. A further important general principle is that these proteins turn over rapidly, as evidenced by the full recovery by the stigma of its adhesive properties within a short time after protease treatment (Stead, Roberts and Dickinson 1980). 

The most likely component of the pollen grain to bind to the stigma in this manner is the tapetal derived pollen coating, trephine. A comparison of the surface components with recognition potential present on the pollen and stigma of G. Gandavensis has revealed the intriguing fact that both factors contain a complex mixture of proteins, glycoproteins, and glycolipids. Thus, the pollen surface molecules apparently complement the components of the stigma to produce an ideal adhesion (Clarke et al 1977, 1979). These observations strongly point to the conclusion that interaction between molecules present on the pollen surface and the stigma papilla is the hallmark of adhesion of compatible pollen grains. Whether incompatible pollen grains adhere to the stigmatic papillae in a molecular sense or mechanically stick to the papillae has remained conjectural. 

Qualitative data collected on pollen grains landing on the stigmatic surface of B. Oleracea after cross and self-pollinations have revealed differences in adhesion due to physicochemical reactions (Roggen 1972). From counts of pollen released from pollinated stigmas of B. Oleracea, it appears that after an initial sluggish period of about two hours, during which time self-pollen grains bind less firmly than cross-pollen, the former adheres to an extent comparable to cross-pollen (Stead, Roberts and Dickinson 1980). These cases are exceptions rather than the rule.

Besides the stigma, other floral organs of Arabidopsis, such as the style, ovary, anther, petal, and sepal, are receptive to pollen grains and support their germination and pollen tube growth (Kandasamy, Nasrallah and Nasrallah 1994). In the fiddlehead (fdh) mutant of Arabidopsis, wild-type pollen grains germinate and grow on a floral part formed by the fusion of non-carpel organs of the shoot (Lolle and Cheung 1993). These observations suggest that components of the pollen recognition system are present in the shoot epidermis and in all floral organs during their ontogeny but are segregated to the surface cells of the stigma and other floral parts by the action of the FDH gene.

Pollen Hydration

Interactions


Hydration by the uptake of water is the first change observed incompatible pollen grains after they make contact with the stigmatic surface; the best evidence establishing this as a recognition event is the fact that, in many plants, foreign pollen grains remain dry on the stigma and are not recognized. The recognition reaction apparently functions by regulating water traffic from the stigmatic cells to individual pollen grains; for example, when pollen grains are placed on the stigmatic papilla in a chain, but with only one pollen grain in contact with the surface of the papilla, the one grain is preferentially hydrated, but the others remain dry (Sarker, Elleman and Dickinson 1988; Hiilskamp et al 1995). 

Apart from the sweeping changes in the cytoplasmic activities of the pollen grain, hydration plays an important role in restoring the structure of the plasma membrane of the vegetative cell. The exact sequence of changes that occur in the plasma membrane upon hydration is not known, but it appears that the partly dissociated membrane acquires normal osmotic properties during controlled hydration (Shivanna and Heslop-Harrison 1981). In most species, the pollen grain absorbs water rapidly from the stigmatic papilla, whereas in others it obtains water from the exudate on the stigma surface. The degree of hydration depends upon the state of the stigma, whether it is wet or dry, and the state of dryness in which the pollen grains are held prior to pollination (Gilissen 1977; Stead, Roberts and Dickinson 1980; Barnabds and Fridvalszky 1984; Zuberi and Dickinson 1985). 

We also need to know about the substances from the exudate taken up by the pollen grains. Autoradiographic studies have shown that immature pistils of Primula Officinalis and Ruscus aculeatus cultured on an agar medium containing 45 Ca 2+ transport the ion through the stigmatic papillae to germinating pollen grains; the implication of this observation is that any Ca 2+ present in the stigmatic exudate might be similarly taken up by pollen grains germinating in vivo (Bednarska 1991). Little information is available on the dynamics of pollen hydration on wet stigmas as compared to that on dry stigmas. One reason for this limited knowledge is that the matrix of the exudate provides the pollen grains with an instant source of water at controlled osmotic pressure for hydration without recourse to other physical processes.

Hydration of pollen grains alighting on dry stigmas is apparently a complex process because dry stigmas offer a less hospitable surface than wet stigmas. A few cases will be considered in which events occurring on the stigmatic papillae and on the pollen grains have been followed after compatible pollinations of dry stigmas. The grasses are unusual among the species so far investigated in showing extremely rapid initial exchanges between pollen and stigma. A study of pollen hydration in Secale cereale has been particularly illuminating in showing by time-lapse microphotography three distinct phases in the hydrodynamics of the pollen grain immediately preceding germination. 

The first phase is the passage of water from the stigmatic papilla into the pollen grain. Although this influx greatly increases the water content primarily in the vegetative cell, some subtle changes are also initiated on the plasma membrane of the cell until it is re-formed. Continued uptake of water by the pollen leads to the second phase, which begins before the plasma membrane becomes an effective osmotic barrier. During this phase, rather than expanding to the point of bursting, the pollen grain begins to lose water and solutes by exudation from the germination pore and through the micropores of the exine. Proteins and other mobile constituents held in the pollen wall are also probably leached out from the surface of the pollen grain onto the stigmatic surface. 

The use of fluorescent-labeled pollen wall proteins seems to confirm that during pollination, exine-held proteins might indeed penetrate the cells of the stigma. The third phase, during which the plasma membrane is completely reconstituted, is the most critical, and further traffic of water molecules during this period is determined by the rules governing an osmotically active plant cell. With the completion of this sequence, the pollen grain has built up considerable hydrostatic pressure which will be relieved by the emergence of a pollen tube (Figure 7.9). By the most generous estimates, these events resulting in germination do not take more than two minutes from the time the pollen makes contact with the stigmatic papilla (J. Heslop-Harrison 1979a; Vithanage and Heslop Harrison 1979). Although details of pollen-stigma interaction are scanty for other species of grasses studied, they also seem to follow the same general principles as in rye (J. Heslop-Harrison 1979b, c; J. Heslop-Harrison and Heslop-Harrison 1981).

Special attention has been focused on the interpolated period of efflux from the pollen grain, which includes intine-held proteins in grasses like Phalaris tuberosa (Knox and Heslop-Harrison 1971c). A rapid release of pollen-held proteins such as anti- gen-E accounts for the presence of a fluid that coats the pollen grains of Ambrosia trifida and Cosmos bipinnatus after they land on their respective stigmatic papillae (Knox 1973).

It has proved difficult to observe pollination related changes in pollen grains through the use of conventional fixatives without inducing artifactual hydration. Anhydrous fixation techniques have revealed that several structural changes in the exine coating accompany hydration of the pollen grain of Brassica oleracea. Along with water, some materials emitted from the stigma and loaded into the pollen grain account for these changes. The first event that is consequent upon the pollen grains making contact with the stigma is the movement of the exine coating to form an appressoria-like "foot" on the surface of the papilla.

The control of this traffic is poorly understood, but there is no doubt that it is linked in some way to the occurrence of recognition reactions at the interface between the pollen grain and the stigma. Progressive hydration elicits structural changes in the coating, which becomes electron opaque due to densely packed membranous assemblies (Dickinson and Elleman 1985; Elleman and Dickinson 1986, 1990). Transfer of exine-held materials of the pollen onto the stigma surface has also been noted following pollination in other plants with dry stigmas (Elleman, Franklin-Tong and Dickinson 1992).

Although these examples serve to illustrate the physical and structural aspects of pollen hydration, they do not address the molecular nature of pollen-stigma communication. Contact with a compatible stigma triggers the synthesis of a particular set of proteins in the pollen grains of Brassica napus. Because some of these proteins are phosphorylated, a conceptual pattern of signaling mechanism during compatible pollinations that involves protein phosphorylation may be in prospect (Hiscock, Doughty and Dickinson 1995). To investigate the signaling mechanism during pollen-stigma interaction from a different angle, Preuss et al (1993) isolated mutants of Arabidopsis that affect pollen-stigma communication. Although wild-type pollen grains hydrate rapidly on the stigma and germinate, those of the mutant defective in pollen-pistil interactions (pop-1) does not absorb water from the stigma and consequently fail to germinate. 

A note of interest in linking the loss of germinability to the failure of pollen hydration is the observation that pop-1 pollen grains are rescued in vivo on the stigma under humid growth conditions or in vitro by culture in a simple nutrient medium. A connection between pollen hydration and long-chain lipid molecules as indicated by the absence of wax on the stem of pop-1 plants. Chemical analysis showed that wax deficiency on the mutant stem is correlated with a deficiency in long-chain lipids on the mutant pollen grains, and electron microscopy confirmed that mutant pollen grains lack lipidic trephine on their exine coating. A model for pollen hydration in Arabidopsis suggested by these results is that during compatible pollination, the pollen grain signals the stigma surface and establishes communication with the help of lipid and trephine molecules; this initial molecular interaction evidently sets the stage for the cascade of events beginning with the uptake of water.

Analysis of other mutants that affect pollen hydration on the stigma of Arabidopsis has also focused on the lipid product of the pollen wall as a major player in the binding of the pollen grain to the stigmatic papilla (Aarts et al 1995; Hiilskamp et al 1995). Work with mutant pollen grains of Petunia sp. deficient in flavonols has revealed a signaling role for kaempferol in pollen germination on the stigma. This is based on the finding that mutant pollen grains can be rescued by germinating on wild-type stigma or in micromolar quantities of kaempferol isolated from the stigma (Mo, Nagel and Taylor 1992). A different scenario is noted in transgenic plants of P. Hybrida characterized by the absence of flavonols in the pollen grains. Plants are rendered self-sterile by this manipulation and, although pollen grains germinate normally in vitro, their growth is stymied after a short period (Ylstra et al 1994).

Stigmatic Response to Pollination


During pollen-stigma interactions, changes occurring on the stigmatic papillae are the very epitome of cell recognition events on the female side. Biological changes associated with these interactions are reflected in physical changes, such as voltage variations in the style specific for compatible pollination (Spanjers 1978). As seen earlier, the morphogenesis of the papilla is the major event in the development of the stigma. Cell ablation techniques have been used to determine the extent to which full development of the papilla is necessary for normal interaction with compatible pollen grains. 

Wild-type pollen grains of Arabidopsis germinate and form tubes on the surface of stigmatic papillae that are stunted in growth by the introduction of DT-A toxic gene fusion. This shows that the pollen recognition mechanism functions even in flowers with stigmatic cells that, in contrast to cells in the normal stigma, are not biosynthetically fully active (Thorsness et al 1993). However, as shown in transgenic tobacco engineered by the introduction of the BARNASE gene, if the toxic gene wipes out the stigmatic papillae completely, pollen germination is reduced and the pollen tubes fail to penetrate the transmitting tissue of the style. The block to pollen germination and pollen tube growth is overcome by bathing the ablated surface in exudate from the wild-type stigma (Goldman, Goldberg and Mariani 1994). Evidently, in ways unknown, the exudate provides conditions for pollen-stigma interactions even in the absence of the stigmatic cells.

During pollen-stigma interactions, changes occurring on the stigmatic papillae are the very epitome of cell recognition events on the female side. Biological changes associated with these interactions are reflected in physical changes, such as voltage variations in the style specific for compatible pollination (Spanjers 1978). As seen earlier, the morphogenesis of the papilla is the major event in the development of the stigma. 

Cell ablation techniques have been used to determine the extent to which full development of the papilla is necessary for normal interaction with compatible pollen grains. Wild-type pollen grains of Arabidopsis germinate and form tubes on the surface of stigmatic papillae that are stunted in growth by the introduction of DT-A toxic gene fusion. This shows that the pollen recognition mechanism functions even in flowers with stigmatic cells that, in contrast to cells in the normal stigma, are not biosynthetically fully active (Thorsness et al 1993). 

However, as shown in transgenic tobacco engineered by the introduction of the BARNASE gene, if the toxic gene wipes out the stigmatic papillae completely, pollen germination is reduced and the pollen tubes fail to penetrate the transmitting tissue of the style. The block to pollen germination and pollen tube growth is overcome by bathing the ablated surface in exudate from the wild-type stigma (Goldman, Goldberg and Mariani 1994). Evidently, in ways unknown, the exudate provides conditions for pollen-stigma interactions even in the absence of the stigmatic cells.

Pollen Germination and Pollen Tube Growth


Following hydration on the stigma, some characteristic changes directly concerned with germination take place in the cytoplasmic domain of the pollen grain. Within a few minutes after hydration, the pollen cytoplasm appears quite different from the dehydrated pollen cytoplasm. In Brassica oleracea, the dry pollen grain is characterized by the presence of many spherical fibrillar bodies at the periphery of the protoplast, whereas the protoplast of the hydrated pollen is conspicuously stratified and contains a peripheral layer of membranous cisternae (Elleman and Dickinson 1986).

Growth of pollen tubes and their entry into the stigma after compatible pollination have been described in several plants. However, we face serious problems if our aim is to build a unified picture of the patterns observed. Pollen tube growth in Crocus chrysanthus fits in well with a model in which the tip of the tube enters the stigmatic papilla after lysis of the cuticle by cutinase. Thereafter, the pollen tube continues to cruise toward the ovary by growing beneath the cuticle close to the underlying pecto-cellulosic wall. From the fact that pollen tubes do not penetrate the cuticle when the proteins of the stigma exudate are degraded enzymatically, lysis of the cuticle by pollen-held cutinase without cooperation from the stigmatic exudate appears unlikely. 

Rather, it has been suggested that the pollen grain contributes a precursor of the enzyme, which is activated by a factor present on the stigmatic surface (J. Heslop-Harrison and Heslop-Harrison 1975, 1981; Y. Heslop-Harrison 1977). Once pollen germination has begun on the stigma of barley, penetration of the stigmatic hair by the pollen tube is sine qua non for its subsequent growth, as it is not unusual to see penetration of the hair by short tubes, random growth of tubes in the air before penetrating the hair, or pollen tubes bypassing one hair before penetrating another (Cass and Peteya 1979). 

Germination of pollen grains on the stigma of Gladiolus gandavensis is followed by pollen tube penetration of the cuticle and continued growth of the tube over the underlying pectocellulosic layer. Growth of the pollen tube into the style and through the stylar canal toward the ovary occurs in a matrix of mucilage that is secreted by the stigma and serves as a guide (Clarke et al 1977a). 

In Lychnis alba (Caryophyllaceae) (Crang 1966), in members of Asteraceae such as Ambrosia tenuifolia and Helianthus annuus (Knox 1973; Vithanage and Knox 1977), and in members of Brassicaceae such as Arabidopsis, Brassica oleracea, Diplotaxis tenuifolia, Raphanus raphanistrum, and R. sativus (Kroh and Munting 1967; Dickinson and Lewis 1973a; Hill and Lord 1987; Elleman et al 1988; Elleman, Franklin-Tong and Dickinson 1992), pollen tubes penetrate the cuticle and travel between the two wall layers of the stigma cells to the base of the papilla, where they enter the middle lamella of the transmitting tissue. Unlike in C. chrysanthus, in the other examples cited, a role for pollen-held cutinase in the penetration of the pollen tube appears likely but has not been established. 

Pollen-stigma interaction in Vicia faba is somewhat unusual, since pollen tubes enter the vicinity of the stigmatic papillae through torn gaps in the thick layer of cuticle, thus circumventing the need for enzymatic disruption of cutin (Lord and Heslop-Harrison 1984). Cutinase action also appears unlikely in Gossypium hirsutum (Jensen and Fisher 1969) and Oenothera organogenesis (Dickinson and Lawson 1975). In the former, the pollen tube grows downward by riding on the surface of the papillae and penetrates neither the cuticle nor the cell wall until it reaches the transmitting tissue of the style. In O. organogenesis, the pollen tube grows through the middle lamella of adjacent cells of the stigmatic papillae and travels down the surface of the cells of the subjacent layer (alveolar tissue). Of course, the concept of pollen tube growth at this critical juncture in the absence of interaction with the cells of the stigma is a gross oversimplification in terms of recognition reactions.

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