Pistil Factors Controlling Pollination

The successful establishment of angiosperms on land is in part determined by their floral design. Because plants cannot move to find the ideal mate, they have developed a great variety of flowers to provide different mechanisms of pollen release, pollen transfer, and deposition of the pollen from the male to the female sexual organs, the anther and the pistil, respectively. Pollination ensures the maintenance of the species, but it is also a means to increase genetic diversity and, with it, the potential to adapt to new environments. The position and morphology of the anthers and the pistil have often coevolved with the mode of pollen dispersal and pollen receipt, aided either by wind or by animals. Nevertheless, pollination can fail at various points during these processes, causing the extinction of rare plants and lower crop yields (Wilcock and Neiland, 2002). The pistil, the pollen-accepting organ that occupies the central position in a flower, is composed of one or more fused carpels that bear the ovules. Pistil development initiates with the formation of the carpel primordia, and the floral identity genes of class C, such as AGAMOUS in Arabidopsis thaliana and PLENA in Antirrhinum majus, dictate carpel identity (Ng and Yanofsky, 2000). Carpel fusion occurs very early in pistil development. Even in species with single pistils, fusion of the carpel margins is required to form a closed carpel. For instance, tobacco (Nicotiana tabacum) and Arabidopsis pistils are made from the fusion of two carpels, and that of the cultivated tomato is formed from five fused carpels (Gasser and Robinson, 1993). Close to the time of ovary closure, the carpel walls extend vertically to form one or more hollow cylinders, the styles. This process requires cell division at first and cell elongation later. The length, number, and structure of the styles are typical within each species and variable between them. Stylar extension facilitates pollen capture, and the wide variety of pistil morphologies reflects the different pollination mechanisms found among the angiosperms (Barrett et al., 2000). While the style is elongating, the inner tissues differentiate to form the specialized secretory zone of the stigma on the top of the style and the transmitting tissue within the hollow cylinder of the style. In some species, such as lily, the style remains hollow, with only one layer of secretory tissue lining the inner surface of the cylinder (Dickinson et al., 1982). At flower maturity, when pollination takes place, the pistil is fully developed and composed of stigma, style, and ovary. Whether the pollen is transported by the wind or by animal pollinators, after landing on the stigma the pollen grain hydrates and germinates a tube. This tube then penetrates the specialized tissues of the pistil, growing into the stigma and the style to reach the ovules in the ovary. During this process, numerous cell–cell interaction events occur between the cells of the sporophyte (the pistil) and the male gametophyte (the pollen grain and the tube). Most of the existing knowledge regarding pollen–pistil interaction was gathered from the study of self-incompatibility in several species. Nevertheless, considerable data are now available on the prepollination and postpollination events and molecules that make the pistil ready to interact with the pollen after compatible pollinations (Lord and Russell, 2002). Some of these interactions are discussed in this review. Stigma receptivity to pollen can persist from 1 h to several days in different species (Heslop-Harrison, 2000) and is influenced by several factors. Whether it is a dry stigma or a wet stigma, receptivity is defined as the ability to “capture” pollen by adhesion, to let it hydrate and consequently germinate a pollen tube. The appropriate stage of stigma development is crucial for receptivity. For example, on immature stigmas of pear flowers, mature pollen can adhere but do not hydrate and germinate. On a degenerating stigma, pollen can adhere, hydrate, and germinate, but pollen tube growth arrests abruptly (Sanzol et al., 2003). A cuticle of varying thickness is deposited on most stigmas above the epidermis, and compatible pollen deposited on a heavily cutinized surface never germinates (Heslop-Harrison, 2000). At the receptive stage, the cuticle breaks at some places, aided either by visiting insects or by increasing turgidity of the stigma, and the activity of some enzymes such as esterases increases in the stigma of several species (Dafni and Maues, 1998). The pollen of Brassica napus was shown to carry an active cutinase to break the cuticle of the papilla (Hiscock et al., 1994; Edlund et al., 2004). The epidermis of the stigma differentiates to form the specialized papilla cells. Crucifers, such as Brassica species and Arabidopsis, have a dry stigma with many large papillae that interact directly with the pollen. Here, the pollen is accepted if compatible, or rejected if incompatible or from a foreign species, and adheres and hydrates. The best-characterized pollen–pistil interaction on a dry stigma is the self-incompatibility response in Brassica. Its components constitute the male determinant S-locus cystein rich protein, the S-locus glycoprotein female determinant secreted by the stigma into the cell wall, the S-locus receptor kinase located in the stigmatic plasma membrane, and its target arm-repeat-containing protein 1, also produced in the stigma (Nasrallah, 2000). However, little is known about the genes required for successful (compatible) pollen–stigma interaction in crucifers. No mutants have been identified to date with defects in papillar cell functions, although cell ablation experiments clearly demonstrated that papillae are required for pollination. Papillar cell ablation, in combination with differential display, also was used to identify genes expressed in the Brassica napus epidermis (Kang and Nasrallah, 2001). The results of this experiment led to the identification of the PIS63 gene, which is homologous with an Arabidopsis gene of unknown function. By a transgenic approach, it was shown that a reduction of PIS63 expression in the stigma correlates with reduced pollen germination and seed set, but pollen adhesion was not affected (Kang and Nasrallah, 2001). This finding, together with the identification of Arabidopsis pollen mutants affected in adhesion (Edlund et al., 2004), supports the idea that pollen adhesion on a dry stigma is a property of the pollen. The adhesion of pollen on wet stigma is facilitated by the presence of the exudate, which can be aqueous, as in lily, or lipidic, as in tobacco and petunia. In addition, proteins and sugars are present in both types of exudate. Pollen hydration and tube germination can occur very fast or may take up to 1 h, depending on the degree of pollen desiccation at the time of anther dehiscence. Grass pollen, for example, is never fully dehydrated and is metabolically active when shed from the anthers. This makes it very vulnerable, but it germinates within minutes after landing on a stigma. By contrast, pollen of lily is very dehydrated when released, which enables it to survive under extreme environmental conditions, but it takes ∼1 h to germinate after hydration on the appropriate stigma (Heslop-Harrison, 2000). Hydration on the dry stigmas of the crucifers requires contact with the papillae and is aided by the pollen coat that flows from the exine to form a contact zone between the two (Elleman et al., 1992). In particular, lipids and oleosin-like proteins on the pollen coat were shown to be essential for hydration in Arabidopsis (Edlund et al., 2004), demonstrating that, as in pollen adhesion, hydration also is determined by the pollen. We found no oleosin-like proteins on the coat of tobacco pollen, suggesting that on this wet stigma hydration may be facilitated by other factors (Bots and Mariani, 2004). In tobacco and other solanaceous species, the lipidic exudate is produced in the cells of the secretory zone of the stigma and is secreted at pistil maturity. Underneath the exudate, a thin layer of water (in the form of crystals) surrounds the cells of the secretory zone. After pollination, pollen grains sink through the exudate and establish direct contact with the stigma or with each other. Using cryo-scanning electron microscopy, it was evident that at the contact site between pollen and stigma, the water crystals gradually disappeared (Wolters-Arts et al., 2002). We produced transgenic tobacco plants in which the secretory zone of the stigma was ablated by tissue-specific expression of a Barnase ribonuclease. Pollen–pistil interaction is arrested in this plant, resulting in female sterility (Goldman et al., 1994). In particular, this stigmaless pistil does not produce exudate, and cryo-scanning electron microscopy images confirmed that no water crystals were present around the dead cells of the ablated stigma (Wolters-Arts et al., 2002). Yet, upon application of exogenous tobacco exudate or a mixture of triacylglycerides, pollen still could hydrate. It is not clear how the water passes through the dead cells of the stigmaless pistil to the pollen grain. However, contact alone is not sufficient for hydration, because even the exertion of a constant mechanical pressure on the pollen grains did not lead to hydration without exudate or lipids (Wolters-Arts et al., 2002). Recently, many water channel proteins, collectively named aquaporins, were described in plants; these indicate the existence of a very rapid and regulated water transport across biological membranes (Johanson et al., 2001). Some of these aquaporins or aquaporin-like genes are expressed in the pistil, among other plant tissues, of Solanum chacoense (O'Brien et al., 2002) and Brassica (Marin-Olivier et al., 2000; Dixit et al., 2001). Although it is attractive to speculate that they play a role in pollen hydration by the stigma, 35 putative aquaporin genes, classified on the basis of sequence identity, were found in the genome of Arabidopsis. With such high redundancy, it may be difficult to prove the function of aquaporins in the pollen–stigma interaction. We have produced transgenic tobacco plants in which a class of PIP2 aquaporins, expressed in flower organs, was silenced by RNA interference. We found no effects on pollen hydration, pollen tube growth, and seed set (M. Bots and C. Mariani, unpublished data). Once pollen is hydrated and germination occurs, tube growth is directed within the stigma. In dry stigmas, tube growth occurs through the papilla cell wall, and pollen tube penetration is accompanied by cell wall expansion or loosening in the stigma (Elleman et al., 1992). Tube growth seems to be facilitated by wall-degrading enzymes produced either by the pollen itself or by the stigma. This is in agreement with the finding that group I allergens of grass pollen have expansin activity (Cosgrove et al., 1997) and that a polygalacturonase was localized in Brassica germinating pollen (Dearnaley and Daggard, 2001, and references therein). In tobacco, a species with a wet stigma and a solid style, pollen tubes grow through the intercellular spaces between the cells of the secretory zone, within the exudate produced by these cells. Interestingly, we recently discovered that tobacco exudate has cell wall–loosening activity (J. Nieuwland, J. Derksen, C. Hilbers, and C. Mariani, unpublished results). The pistil pollen allergen-like (PPAL) protein, one of the proteins secreted in the exudate, is highly similar to β-expansins (Pezzotti et al., 2002) and obviously was the most suitable candidate for a cell wall–loosening activity that could facilitate pollen tube growth in tobacco stigmas. However, we found that PPAL is not active in cell wall loosening in an in vitro assay using an expansometer constructed according to Cosgrove (1989). Moreover, we have determined that another protein is responsible for cell wall–loosening activity. This protein is a lipid transfer protein (LTP), the most abundant protein in the exudate of tobacco (J. Nieuwland J. Derksen, C. Hilbers, and C. Mariani, unpublished results). LTPs can bind acyl lipids in vitro (Kader, 1996) and constitute the largest group in the catalog of Arabidopsis genes involved in acyl lipid metabolism (Beisson et al., 2003). LTPs represent a group of proteins whose sequences are highly divergent and that may have different in from another function for proteins is that of the protein secreted in the lily exudate 2003). has a hollow stigma and a style with a secretory epidermis that pollen tubes in to the ovary. no tissue penetration is required for pollen tubes to grow from the stigma into the style. with another stigma protein, activity on lily pollen tubes in vitro J. and unpublished data). Pollen tubes their growth a of and this activity. Using stigmaless transgenic tobacco plants (Goldman et al., we pollen–pistil interaction in this to the components The ablated surface of this stigmaless pistil is and when tobacco pollen is used for pollination, it to hydrate. is pollen hydrates but only a pollen tubes germinate. This finding that the is only on the stigma and not in the pollen. The of germination and tube but penetration of the pistil tissues when we exudate from pistils, pollen tubes and directly in the pistil tissues (Goldman et al., 1994). we are the function of the proteins secreted in the exudate, some of which and have been discussed of of at h after After application of exudate, pollen grains on the stigmaless surface hydrated and germinate and the pollen tubes the to of the application of lily exudate and are pollen pollen transmitting function to LTPs and to other proteins secreted in the exudate is that of et al., et al., proteins, and other proteins and references were shown to be expressed and to in the stigmas of species of the to or However, these are not directly to pollination. that can be regarding pollen–stigma interaction is that although pollination on dry stigmas and wet stigmas very are in the two For example, lipids are required in crucifers and in solanaceous species for pollen hydration and tube and proteins such as have been to pollen tube growth in both plant The only in these factors the pollen coat in species with dry stigmas and the exudate in species with wet stigmas on styles can be classified as or In of many such as lily, pollen tubes grow in the with the transmitting Most such as species, have a closed style in which the pollen tubes grow through the transmitting a with the stigma secretory zone. The cells of the transmitting tissue are in a by on their their walls are by the intercellular that they has shown that the proteins, and to function in pollen tube and In this in pollen tubes grow with considerable pollen for instance, take to h to reach the at a of from the stigma. This makes the of pollen tube growth Pollen of many species can germinate and grow a tube in vitro in a that at and an However, pollen tubes in vitro never reach the they can when growing in the pistil 2003). Nevertheless, the with which tube growth can be in vitro has been very in the study of pollen tube cell et al., 2001). 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However, pollen tubes do not to a in other molecules be involved in this By the described in the pollen–pistil interaction is the response in Brassica. However, very are in which molecules play a crucial and have demonstrated that the two tomato and from pollen and when expressed The in pollen after pollen tube germination in but it if is to the germination of the is by the of A for in that a (the in a to protein to and it, and the in the pollen tube et al., 2004). The of this in pollen tube growth is still at the the for the growing pollen tube is In some pollen tubes around on the surface by produced and by specialized such as the 2001). as little the the and to pollen tube growth at that In some species, pollen tubes if the is not active in the of 2001). The of the pollen tube to the was shown recently to on the female A of Arabidopsis mutants in development or function also were found to be to and pollen tubes et al., et al., and 2000; et al., et al., 2004). an study using cell ablation on ovules of has shown that the for pollen is a by the cells the cells et al., et al., 2004). these data no that the pistil molecules for and for pollen tube However, many of these molecules to be and present in the pistil pollination occurs, that the pistil is to pollen tube This also is by the that, to only a genes have been found to be by pollination, such as in and although is for gene expression by this factors also are produced in the style and were in pollen tube However, these often are in one species, which makes it difficult to this only events in species have been In many different types of pollen are present and may be transported by wind or on a pistil that is not of the species. the many plants have developed that in the pistil either pollen tube growth, or after causing of the The pistil, an role in the of gene between species and in the maintenance of the species. 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