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Micropylar pollen tube guidance and burst: adapted from defense mechanisms?
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Micropylar pollen tube guidance and burst: adapted from defense mechanisms? Thomas Dresselhaus and Mihaela L Ma´rton After the first description of fertilization in flowering plants some 125 years ago (Strasburger E: Neue-Untersuchungen u¨ber den Befruchtungsvorgang bei den Phanerogamen als Grundlage fu¨r eine Theorie der Zeugung. Gustav Fischer; 1884), we are finally beginning to understand the various molecular mechanisms leading to sperm delivery and discharge inside the hidden micropylar region of the female gametophyte (embryo sac). The last phase of pollen tube guidance culminating in tube burst and explosive release of tube contents requires extensive crosstalk between both male and female gametophytes. The first molecules identified that play key roles in these processes represent highly polymorphic proteins, similar to major components of the plant innate immune system. Here we summarize recent advances and briefly discuss the underlying molecular mechanisms also in respect to prezygotic barriers of reproductive isolation. Address Cell Biology and Plant Physiology, University of Regensburg, 93053 Regensburg, Germany Corresponding author: Dresselhaus, Thomas ([email protected])
Current Opinion in Plant Biology 2009, 12:773–780 This review comes from a themed issue on Cell Biology Edited by Jirˇı´ Friml and Karin Schumacher Available online 4th November 2009 1369-5266/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2009.09.015
Introduction During the evolution of flowering plants, their sperm cells have lost mobility and are delivered to the female gametes, egg, and central cell, via the vegetative pollen tube cell to achieve double fertilization. The journey of the two sperms cells via the pollen tube, from its germination site at the papillae cells of the stigma, through the transmitting tract inside the style toward the target female gametophyte (embryo sac) is assisted by intensive communication between the tube cell and surrounding sporophytic maternal tissues. The past two decades have seen tremendous progress in our understanding of the underlying molecular mechanisms of pollen tube germination and growth, sporophytic guidance as well as various self-incompatibility systems occurring between www.sciencedirect.com
haploid pollen/pollen tube and diploid cells of the pistil. Excellent reviews have been published [1–4] and these processes will not be further considered here. In contrast, until recently there was little progress in elucidating the molecular mechanisms of the femalegametophyte-controlled last phases of pollen tube growth and guidance, growth arrest, sperm discharge and transport as well as gamete fusion. This is due to the fact that the female gametophyte, also referred to as the ‘hidden generation’ [5], is deeply embedded in the maternal tissues of the ovary and ovule. However, the characterization of novel mutants [6,7,8,9,10], reverse genetic approaches [11,12,13,14], in vitro methods [12,15], and the development of various gamete-specific markers as well as techniques such as spinning disk live-time imaging [16,17] finally enabled the visualization of the cellular processes preceding plant gamete fusion and the establishment of appropriate first mechanistic models. We will therefore restrict our review on micropylar guidance, and inter-gametophytic signaling preceding pollen tube growth arrest and burst, and comment on the evolution of some key molecules involved that might contribute to prezygotic reproductive isolation barriers in plants [18–20].
Gametophytic pollen tube attraction After emerging from the transmitting tissue, the male gametophyte (pollen tube) is thought to be guided by chemotactic factors secreted by the synergid cells of the female gametophyte. This hypothesis is supported by genetic, cell ablation, and in vitro investigations, using excised Arabidopsis thaliana and Torenia fournieri ovules, showing that short-range pollen tube guidance is controlled by the female gametophyte via species-specific chemotactic signals [15,21–24]. Taking advantage of the naked embryo sac of T. fournieri, laser-assisted cell ablation experiments showed that the two synergid cells are necessary and sufficient to attract the pollen tube. Egg and central cells are either not involved in pollen tube attraction in T. fournieri, or not able to produce/secrete sufficient amounts of the guidance molecule. In maize, it seems that the egg cell contributes to the synthesis of the attraction signal [11], and in Arabidopsis even the central cell was suggested to play a critical role in pollen tube attraction [25]. However, it cannot be ruled out that the corresponding CCG (CENTRAL CELL GUIDANCE) gene encoding a potential transcriptional regulator acts rather indirect and is instead required, for example, for embryo sac maintenance. Current Opinion in Plant Biology 2009, 12:773–780
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The maximum distance of attraction in vitro is about 200 mm in T. fournieri [2], and about 100 mm in Arabidopsis [14,15] and maize (A Lausser et al. [26], Sporophytic control of pollen tube growth and guidance in maize, unpublished data), which contradicts models of longrange guidance by the female gametophyte [27]. As estimated mathematically, the maximum distance of gametophyte attraction is strongly limited by its production and not expected to significantly exceed this distance [22]. Mature synergid cells contain large numbers of secretory organelles such as stacked arrays of endoplasmic reticulum, Golgi complexes as well as vesicles in a secretory zone in proximity to the micropylar filiform apparatus [22,28–30]. This extracellular structure is generated by cell wall invaginations of both synergids and is composed mainly of cellulose, hemicellulose, and glycoproteins. It is thus likely that this apoplastic structure can take up and distribute vesicle contents secreted by the synergid cells. In conclusion, we suggest to consider the synergid cells as the ‘glandular cells’ of the female gametophyte similar to follicle cells in mammals [31]. Owing to the occurrence of high Ca2+ concentrations in the synergid cells, Ca2+ has long been considered a potential attractant [32]. But elevation of the Ca2+ ion concentration in in vitro pollination assays using the above described T. fournieri system did not affect the observed attraction [21], indicating that it does not serve as an attractant at all or is only part of an attractant cocktail of molecules. A gamma-amino butyric acid (GABA) gradient that is established along the pollen tube pathway was also suggested to guide the tube toward the micropylar region of the ovule [33]. However, in vitro pollen tube attraction studies using synthetic GABA have not been performed to demonstrate that it indeed functions as a pollen tube attractant. Moreover, various in vitro studies have indicated that pollen tube guidance and sperm release depends on species-specific factors rather than on general molecules such as Ca2+ or GABA. The first gene identified that encoded a candidate micropylar attractant of the pollen tube secreted by the female gametophyte was ZmEA1 (Zea mays EGG APPARATUS1), which is specifically expressed in the egg apparatus (egg and synergid cells) of maize and downregulated after fertilization. ZmEA1 encodes a polymorphic precursor protein of 94 amino acids and was shown to be secreted to the cell walls of micropylar nucellus cells of mature embryo sacs. Pollen tubes arrived at the micropyle of ZmEA1 knockdown plants without penetrating the intercellular space of micropylar nucellus cells providing further support that ZmEA1 might be involved in micropylar pollen tube guidance [11]. However, a biochemical proof was still lacking and we therefore show here (Figure 1a and b) that an N-terminal cleaved predicted mature ZmEA1 protein of 49 amino acids can directly attract maize pollen tubes in vitro at a concentration of <10 mM. Secreted cysteine-rich proteins (CRPs) named Current Opinion in Plant Biology 2009, 12:773–780
Figure 1
In vitro pollen tube attraction and burst studies using recombinant or chemically synthesized proteins. (a) 10 mM ZmEA1 was mixed with Alexa Fluor 488 and released in the proximity (ca. 30 mm) of an active maize pollen tube using a micro-capillary. (b) Around 22 min after application, pollen tube tip growth was reoriented toward the region of ZmEA1 droplet release (asterisk). (c) LURE1 (TfCRP1) and Alexa Fluor 488containing gelatin beads (asterisk) were placed about 50 mm from the growing pollen tube tip (arrowhead). Around 20 min after application, the pollen tube tip (double arrowheads) changed growth direction toward the LURE1-containing bead. (d) Addition of ZmES4 induced pollen tube burst at the very tube tip (arrow) and explosive discharge of its contents within <1 min. Scale bars: 50 mm.
LUREs have been recently identified as female gametophyte attractants in T. fournieri [12]. These polymorphic proteins are specifically synthesized in synergid cells and belong to a subgroup of the defensin-like (DEFL) super gene family of CRPs. Immunolocalization showed that LURE1 and LURE2 are secreted to the filiform apparatus. Recombinant mature proteins can attract competent pollen tubes at nanomolar concentrations in vitro in a species-specific manner (Figure 1c). Microinjection experiments of LURE morpholino antisense oligomers into mature embryo sacs significantly impaired pollen tube attraction providing further evidence that the DEFL genes LURE1 and LURE2 encode micropylar pollen tube attractants derived from the synergids. Results from the description of the GEX3 (GAMETE EXPRESSED3) gene of Arabidopsis for a plasma-membrane-located protein containing four PQQ (b propeller) domains [34] are difficult to interpret. Although GEX3 is most strongly expressed in pollen and according to promoter-marker expression at a low level in egg cells, reciprocal crossing experiments using transgenic lines indicate that the observed defect of micropylar pollen tube guidance is caused by the female gametophyte. Whether the observed phenotype is produced by an www.sciencedirect.com
Micropylar pollen tube guidance and burst Dresselhaus and Ma´rton 775
Figure 2
Hypothetical model showing the cellular and molecular mechanisms involved in micropylar pollen tube attraction and sperm discharge. (a) Vesicles containing pollen tube attractants are steadily secreted from mature synergid cells. After binding to pollen-tube-located receptor(s), pollen tube tip growth is reoriented (large arrowhead) in a concentration-dependent manner toward the synergid cells. While vesicles containing pollen tube ligands are also steadily discharged at the tube tip, secretory vesicles containing pollen tube ‘toxins’ are accumulating in the secretory zone of the synergids, but are not discharged. (b) Pollen tube invades the filiform apparatus (black region) between both synergid cells signaling toward the synergid cells via plasma-membrane-located proteins such as FER/SIR and LRE. Activated synergid cells discharge ‘toxin’-containing vesicles leading to pollen tube growth arrest via modulation/activation of pollen tube receptors/ion channels. NO and ROS play a yet unclear role during this process. (c) Intergametophytic signaling induces cell death in the receptive synergid cell and pollen tube burst after a dramatic change in the micro-environment of the pollen tube tip due to synergid degeneration and ion channel opening. Abbreviations: dSY, degenerated synergid cell; MGU, male germ unit consisting of two sperm cells and vegetative nucleus; PT, pollen tube; pSY, persistent synergid cell; SY, synergid cell; vNC, nucleus of the vegetative tube cell.
egg cell or embryo sac maturation defect is unclear, as egg cell identity was not analyzed. A number of reports have demonstrated that secretion of the attractant(s) and thus micropylar pollen tube guidance occurs only when embryo sacs are fully mature [15,22]. In magatama (maa) mutants of Arabidopsis, for example, an embryo sac maturation defect leads to pollen tube growth on the funicular surface, but not inside the micropyle [23,35] indicating both that first, pollen tube guidance until the last, micropylar phase, is sporophytically controlled and that second, only mature embryo sacs are capable of generating/secreting the micropylar attractant(s). Moreover, mutations in a synergid-expressed gene encoding the transcription factor MYB98 show an immature and less organized filiform apparatus; as a consequence, micropylar pollen tube guidance is affected [14]. It was also observed that after the first pollen tube has successfully released its contents, further attraction significantly decreases and second pollen tubes are very rarely found in the micropyle of wild type ovules of various plant species, despite the persistence of the second synergid cell [11,15,24]. This finding indicates that the attractant(s) are degraded, inactivated or no longer secreted, or that additional pollen tubes are actively repulsed. www.sciencedirect.com
Inter-gametophytic signaling preceding pollen tube growth arrest After arrival at the micropyle, the pollen tube directly enters one of the two synergid cells (the receptive synergid) via the filiform apparatus in plant species such as Arabidopsis or T. fournieri [14,22]. In grasses, the embryo sac is additionally embedded by a few layers of stack-like oriented nucellus cells, which have to be passed before the tube can enter the embryo sac [11,27]. After contact with the receptive synergid cell, the latter initially undergoes programmed cell death in Arabidopsis, maize, and many other plant species [36], followed by pollen tube rupture, which generally occurs within a few minutes [2,9], and discharge of its contents including the two sperm cells to achieve double fertilization (Figure 2). This poorly understood process is referred to as pollen tube reception and relies on extensive cell–cell communication between male and female gametophytes [37] via the pollen tube tip and the synergid cell(s) [38]. In recent years, a few Arabidopsis mutants have been described with impaired sperm cell release. In heterozygous feronia ( fer) [39] and sire`ne (sir) [40] mutants about half of the ovules remained unfertilized, although pollen tubes properly penetrated the micropylar region, but Current Opinion in Plant Biology 2009, 12:773–780
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partially failed to arrest its growth and instead continued to grow inside the embryo sac. Moreover, mutant pollen tubes failed to rupture and did not release sperm cells. Molecular cloning revealed that FER/SIR encodes the same plasma membrane localized receptor-like kinase (RLK) belonging to the CrRLK1L-1 subfamily consisting of 17 kinases [7]. Although FER/SIR is also expressed during vegetative development, its function in ovules is associated with the synergids where the protein seems to be asymmetrically distributed at the surface of the synergid cells accumulating in the plasma-membrane invaginations of the filiform apparatus [7]. It was therefore suggested that FER/SIR perceives unknown signals derived from the pollen tube tip to induce a signaling cascade to arrest pollen tube growth. In addition to the identification of the pollen tube derived ligand(s), it still remains to be shown whether FER/SIR signaling induces the release of a feedback signal to the pollen tube, is involved in proper synergid maturation required to release factors that control pollen tube growth, or induces synergid cell death [38] as a prerequisite of pollen tube growth arrest and burst. The identification and molecular characterization of the Arabidopsis LORELEI (LRE) gene provides further support for the existence of gametophyte signaling or recognition mechanisms preceding pollen tube burst [8]. The LRE mutant, resembling the fer/sir phenotype, encodes a small plant specific CRP probably attached to the plasma membrane via a glucosylphosphatidylinositol (GPI)-anchor. Although the authors did not show subcellular localization of LRE at the synergid surface, the gene is specifically expressed in synergids of mature ovules suggesting that it might accumulate at its cell surface and is involved in signaling or recognition mechanisms required to arrest further pollen tube growth. An unusual mutant impaired in pollen tube reception is amc (abstinence by mutual consent). The fer/sir pollen tube overgrowth phenotype occurs only when amc pollen tubes reach amc female gametophytes [10]. AMC encodes a peroxin with limited homology to PEX13 involved in peroxisomal protein import. Peroxisomal targeting signaldependent protein import into pollen peroxisomes is completely disrupted in an amc null mutation. A number of small general molecules such as reactive oxygen species (ROS) and nitric oxide (NO) are produced in peroxisomes [41] and NO has recently been shown to reorient pollen tube growth in a negative chemotropic manner. Additionally, a role of NO in modulating Ca2+ signaling was proposed [42,43]. Whether NO and other small molecules such as ROS modulate the activity of other factors or are directly required for the gametophyte dialogue to reorient or inhibit further tube growth, to induce synergid cell death or induce sperm release remains to be shown by further experimentation. Current Opinion in Plant Biology 2009, 12:773–780
Pollen tube burst Live time observations of pollen tube burst using the T. fournieri in vitro system showed that the pollen tube tip is located within the filiform apparatus between two intact synergid cells before discharge. It was further shown that the plasma membrane of the receptive synergid ruptures within a second at its tip and discharge is explosive [44]. However, the molecular mechanisms leading to pollen tube burst are unclear. As discussed above, Ca2+ has long been considered a key molecule in the fertilization process in plants. Without doubt Ca2+ plays an important role, for pollen tube tip growth [45] and high concentrations have also been determined to exist in the synergid cells [22,32]. It is thus not a surprise that a pollen tube plasmamembrane Ca2+ pump ACA9 (autoinhibited Ca2+ ATPase 9) is required for normal pollen tube growth and sperm discharge. Mutant aca9 pollen tubes reach the micropyle and embryo sac, cease growth, but fail to burst and release their contents [9]. This finding suggests that pollen tube growth arrest and tube burst are separate processes and that sperm discharge requires Ca2+ signaling. DEFL genes ZmES1-4 specifically expressed in the mature maize embryo sac that are immediately downregulated after fertilization [46] have now been shown to be able to directly induce pollen tube burst within <1 min in vitro (S Amien et al., Defensin-like ZmES4 mediates pollen tube burst in maize via opening of the potassium channel KZM1, unpublished data. This is the first report of a molecular mechanism leading to pollen tube burst. DEFL protein ZmES4 induces pollen tube burst in a species-specific manner in maize. Pollen tube membrane depolarization occurs in vitro after ZmES4 application and is accompanied by opening of the inward rectifying K+ channel KZM1) (Figure 1d). Knockdown of ZmES genes leads to normal micropylar pollen tube attraction, but pollen tube contents are not discharged. In vitro pollen tube growth assays showed that mature ZmES4 leads to pollen tube rupture in a species-specific manner accompanied by membrane depolarization preceding burst. The K+ channel KZM1 was identified as a direct target of ZmES4. Whether ZmES proteins additionally act at other targets such as Ca2+ channels, for example the maize ortholog of Arabidopsis ACA9, similar to animal toxins that can modulate various channels simultaneously [47], and thus induces Ca2+ signaling (see above), should be clarified in future experimentations. As shown in Figure 2 it is not unlikely that conserved, but species-specific FER/SIR, LRE, or Ca2+ inter-gametophyte signaling mechanisms are involved in the release of ZmES4 in maize and orthologous proteincontaining vesicles in other plant species. Before fertilization ZmES4 is visible in the secretory zone of the synergids (Figure 2) and vesicles might be released upon signals received from the invading pollen tube, mechanistically similar to the release of cortical granule contents from fertilized animal egg cells/oocytes [31]. www.sciencedirect.com
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Pathogen defense and gametophyte communication involve similar molecular players Remarkably, ZmES4 as well as LURE1 and LURE2 belong to various subclasses of the same super gene family encoding DEFL-proteins in plants [48,49]. Mature defensins (DEFs) and DEFLs are an ancient class of small cysteine-rich antimicrobial proteins/peptides that play an important role in the innate immune system of plants and invertebrates. Plant DEFs and DEFLs have been identified and purified from several tissues, but transcripts are especially abundant in the reproductive tissues of the flower and mature seed. Many DEFL genes are expressed in female gametophyte cells [12,13,46,50,51]. Genome surveys identified >300 DEFL genes in Arabidopsis and around 130 genes in rice [52]. The majority of genes are clustered in the genome and seem to have evolved by duplication and positive selection [53]. However, until recently plant DEFs/DEFLs were mainly shown to be involved in defense where they act as protein translation inhibitors, a-amylase inhibitors, microbial inhibitors, zinc tolerance mediators, ion channel blockers, or protease inhibitors [49]. Most characterized plant DEFs have antifungal activity that inhibits hyphal elongation, with up to 100% growth inhibition and partial lysis of susceptible fungi [48]. However, the exact mode of action of plant DEFs is not yet entirely unraveled. Some plant DEFs with antifungal inhibitory seem to activate an intracellular signaling pathway (e.g. a mitogen-activated protein kinase, or MAPK, signaling pathway) that changes the structural integrity of fungal membranes and culminates in membrane permeabilization [48,54]. This in turn may result in increased Ca2+ influx, K+ efflux, and reduced fungal growth. Moreover, such intracellular signaling cascades correlate with the induction of ROS and NO generation. ROS excess is known to trigger apoptotic cell death in yeast [48] and might also play a role in synergid cell death. It is possible that signaling molecule/s secreted by the cells of the female gametophyte modulate/s intracellular signaling cascades in pollen tubes to generate an excess of ROS via activated peroxisomes leading almost simultaneously to synergid cell death and pollen tube burst. However, since the above-mentioned AMC gene encoding a peroxin was identified in both plant male and female gametophytes [10], it might also be the other way round, that the pollen tube secretes signaling molecule/s modulating intracellular processes including ROS production in the receptive synergid cell, thus triggering cell death. Beyond their role in defense against microbial infection, additional/novel functions of DEFs/DEFLs were acquired in plant development and reproduction. The first examples shown were that they can inhibit the growth of roots and root hairs in A. thaliana [55] as well as pollen viability and seed production [56]. In addition, www.sciencedirect.com
modulation of DEF2 expression in tomato pleiotropically altered the growth of various organs and interfered with fungal invasion by inhibiting tip growth of Botrytis cinerea. Similar to pollen tube and root hair tip growth, fungal hyphal tip growth is a process controlled by a gradient in cytosolic Ca2+ generated by hyphal tip-localized Ca2+ channels [57]. It was thus suggested that DEF2 may alter Ca2+ transport [56] by blocking Ca2+ channels. In animals, most toxins related to plant DEFs/DEFLs were shown to modulate K+ channels, Na+ channels, or Ca2+-activated K+ channels either as pore blockers or as gating modifiers [47]. Whether ZmES4 involved in pollen tube burst solely acts on K+ channels or additionally on others such as Ca2+ channels, whether the target(s) of LURE1 and LURE2 are ion channels or RLKs, and the identification of the molecular mechanisms of NO and ROS action in gametophyte communication are exciting questions for future research.
Evolutionary aspects of defense and reproduction in plants All living organisms have developed efficient mechanisms to defend themselves against pathogen attack. Plants and animals use various intracellular and membrane receptors for the detection of pathogen-associated molecular patterns, serine/threonine kinases for downstream processing, and MAPKs for signal relay. Similar defense responses are elicited, resulting in the generation of NO and ROS, the production of antimicrobial compounds including DEFs, finally culminating in the hypersensitive response or programmed cell death [48,58]. Owing to the lack of an adaptive immune system, it is likely that ancient plants interacted with a broad range of microbial pathogens with different lifestyles and infection strategies, and dedicated a relatively large portion of their genome to defend against pathogens [53]. DEF genes, for example, have been found in all eukaryotic kingdoms and were proposed to have a common ancestor that arose more than a billion years ago [59]. Additionally, the number of plant plasma-membrane RLKs is multiplied compared with animals [60]. In conclusion, the evolutionary arms race between plants and their attackers probably provided ancient water-plants already with a highly sophisticated pool of polymorphic ligand and receptor proteins involved in defense, hundreds of millions of years before going onto land around 460 million years ago [61]. It is thus likely that this pool of genes, which has evolved through positive selection, was also used as a molecular basis to establish novel species-specific gametophytic communication mechanisms required in land plants. Further support for this hypothesis is provided, for example, by the analysis of the dual role of DEF2 in tomato [56] as well as ZmES4 and FER/SIR activity: ZmES4-overexpressing Arabidopsis plants, for example, recover more quickly from fungal attack and, in contrast to wild type plants, fungal hyphae growth is absent a few weeks after infection (S Amien et al., Defensin-like ES4 Current Opinion in Plant Biology 2009, 12:773–780
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mediated pollen tube rupture in maize accompanies opening of the potassium channel KZM1, unpublished data). Moreover, homozygous fer/sir mutants are resistant to fungal infection (U Grossniklaus and co-workers, unpublished data) indicating first, that these proteins are functional and possess targets both in defense and in reproduction and second, that members of these large families evolved de novo function(s) adapted from defense mechanisms according to novel environmental requirements after going onto land.
The research community has finally established the necessary tools and methods to study these hidden processes and to elucidate the underlying molecular mechanisms and their evolution.
Acknowledgements We are grateful to Tetsuya Higashiyama and Irina Kempel for providing images of LURE1 pollen tube guidance and ZmES4 pollen tube burst, respectively. The German Research Foundation (DFG, grant MA 3976/1-1) is acknowledged for financial support to MLM.
References and recommended reading Conclusions The identification of the first molecular players involved in micropylar pollen tube guidance and burst shed new insights into the evolution and functional diversification of polymorphic proteins involved in plant reproduction. It is noteworthy that the angiosperm female gametophyte expresses many more CRP subgroups [12,13,46,50,51,62] as well as members of other polymorphic gene families [7,11] whose functions are completely unknown. Functional studies remain challenging and are complicated by the fact that most of the genes described above occur as gene families showing strongly overlapping expression patterns. The majority of proteins described above seem to have been adapted from defense mechanisms and their species-specific activity may represent a component of reproductive isolation, essential to speciation [19,20]: interspecific pollination experiments within various plant genera resulted, for example, in the above described fer/sir phenotype. Pollen tube overgrowth in the embryo sac was observed after interspecific crosses of Rhododendron [63], as well as with crosses of various Brassicaceae pollen on Arabidopsis stigmata [7]. When ovules of Scrophulariaceae species related to T. fournieri were cultivated together, pollen tubes were significantly more attracted to synergid cells of the corresponding species [21]. These findings indicate that, similar to micropylar pollen tube guidance and burst, inter-gametophytic communication to arrest pollen tube growth is dependent on speciesspecific signaling mechanisms. These findings therefore provide further molecular support that the mechanisms and driving forces of prezygotic barriers to genetic exchange are stronger than postzygotic barriers. Pollen-expressed genes like the polymorphic DEFL-similar sterility-locus cysteine-rich (SCR/SP11) genes and their stigma-expressed receptor kinases of Brassica rapa, as well as various F-box proteins, have been considered as incompatibility factors involved in sporophytic self-incompatibility as one prezygotic barrier [1]. We discussed various incompatibility factors involved in female gametophytic control of sperm delivery, adding first, micropylar pollen tube guidance; second, pollen tube growth arrest; and third, its burst as further prezygotic barriers. In conclusion, it has never been more exciting to study fertilization mechanisms in plants. Current Opinion in Plant Biology 2009, 12:773–780
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10. Boisson-Dernier A, Frietsch S, Kim TH, Dizon MB, Schroeder JI: The peroxin loss-of-function mutation abstinence by mutual consent disrupts male–female gametophyte recognition. Curr Biol 2008, 18:63-68. This paper points to a role of NO and ROS in inter-gametophyte signaling, eventually culminating in the induction of synergid cell death. 11. Ma´rton ML, Cordts S, Broadhvest J, Dresselhaus T: Micropylar pollen tube guidance by egg apparatus 1 of maize. Science 2005, 307:573-576. 12. Okuda S, Tsutsui H, Shiina K, Sprunck S, Takeuchi H, Yui R, Kasahara RD, Hamamura Y, Mizukami A, Susaki D et al.: Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature 2009, 458:357-361. This outstanding study reports a novel function of DEFL genes in micropylar pollen tube attraction occurring in a species-specific manner. www.sciencedirect.com
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Moreover, a laser-assisted microinjection system for plant cells was developed allowing silencing of gene activity after the injection of small RNA species. 13. Jones-Rhoades MW, Borevitz JO, Preuss D: Genome-wide expression profiling of the Arabidopsis female gametophyte identifies families of small, secreted proteins. PLoS Genet 2007, 3:1848-1861. 14. Kasahara RD, Portereiko MF, Sandaklie-Nikolova L, Rabiger DS, Drews GN: MYB98 is required for pollen tube guidance and synergid cell differentiation in Arabidopsis. Plant Cell 2005, 17:2981-2992. 15. Palanivelu R, Preuss D: Distinct short-range ovule signals attract or repel Arabidopsis thaliana pollen tubes in vitro. BMC Plant Biol 2006, 6:7. 16. Steffen JG, Kang IH, Macfarlane J, Drews GN: Identification of genes expressed in the Arabidopsis female gametophyte. Plant J 2007, 51:281-292. 17. Berger F, Hamamura Y, Ingouff M, Higashiyama T: Double fertilization — caught in the act. Trends Plant Sci 2008, 13:437-443. This review nicely summarizes the establishment of live-cell imaging techniques and the development of various gamete-specific marker lines, which are a prerequisite to study fertilization mechanisms in more detail. 18. Vieira A, Miller DJ: Gamete interaction: is it species-specific? Mol Reprod Dev 2006, 73:1422-1429. 19. Rieseberg LH, Willis JH: Plant speciation. Science 2007, 317:910-914. 20. Widmer A, Lexer C, Cozzolino S: Evolution of reproductive isolation in plants. Heredity 2009, 102:31-38. 21. Higashiyama T, Inatsugi R, Sakamoto S, Sasaki N, Mori T, Kuroiwa H, Nakada T, Nozaki H, Kuroiwa T, Nakano A: Species preferentiality of the pollen tube attractant derived from the synergid cell of Torenia fournieri. Plant Physiol 2006, 142:481-491.
32. Dumas C, Gaude T: Fertilization in plants: is calcium a key player? Semin Cell Dev Biol 2006, 17:244-253. 33. Palanivelu R, Brass L, Edlund AF, Preuss D: Pollen tube growth and guidance is regulated by POP2, an Arabidopsis gene that controls GABA levels. Cell 2003, 114:47-59. 34. Alandete-Saez M, Ron M, McCormick S: GEX3, expressed in the male gametophyte and in the egg cell of Arabidopsis thaliana, is essential for micropylar pollen tube guidance and plays a role during early embryogenesis. Mol Plant 2008, 1:586-598. 35. Shimizu KK, Ito T, Ishiguro S, Okada K: MAA3 (MAGATAMA3) helicase gene is required for female gametophyte development and pollen tube guidance in Arabidopsis thaliana. Plant Cell Physiol 2008, 49:1478-1483. 36. Sandaklie-Nikolova L, Palanivelu R, King EJ, Copenhaver GP, Drews GN: Synergid cell death in Arabidopsis is triggered following direct interaction with the pollen tube. Plant Physiol 2007, 144:1753-1762. There is a long lasting discussion on whether synergid cell death in plants is induced before or during pollen tube arrival. The authors of this report summarize and discuss the current knowledge, and additionally show that synergid cell death in Arabidopsis is not triggered before direct pollen tube interaction. 37. Dresselhaus T: Cell–cell communication during double fertilization. Curr Opin Plant Biol 2006, 9:41-47. 38. Punwani JA, Drews GN: Development and function of the synergid cell. Sex Plant Reprod 2008, 21:7-15. 39. Huck N, Moore JM, Federer M, Grossniklaus U: The Arabidopsis mutant feronia disrupts the female gametophytic control of pollen tube reception. Development 2003, 130:2149-2159. 40. Rotman N, Rozier F, Boavida L, Dumas C, Berger F, Faure JE: Female control of male gamete delivery during fertilization in Arabidopsis thaliana. Curr Biol 2003, 13:432-436. 41. Nyathi Y, Baker A: Plant peroxisomes as a source of signalling molecules. Biochim Biophys Acta 2006, 1763:1478-1495.
22. Higashiyama T: The synergid cell: attractor and acceptor of the pollen tube for double fertilization. J Plant Res 2002, 115:149-160.
42. Prado AM, Porterfield DM, Feijo JA: Nitric oxide is involved in growth regulation and re-orientation of pollen tubes. Development 2004, 131:2707-2714.
23. Shimizu KK, Okada K: Attractive and repulsive interactions between female and male gametophytes in Arabidopsis pollen tube guidance. Development 2000, 127:4511-4518.
43. Prado AM, Colaco R, Moreno N, Silva AC, Feijo JA: Targeting of pollen tubes to ovules is dependent on nitric oxide (NO) signaling. Mol Plant 2008, 1:703-714. NO has been previously shown to be able to reorient pollen tube growth in a negative chemotropic manner. This study points to a role of NO and possibly ROS in modulating the activity of other factors, including Ca2+ signaling required for pollen tube tip growth and the gametophyte dialogue.
24. Higashiyama T, Yabe S, Sasaki N, Nishimura Y, Miyagishima S, Kuroiwa H, Kuroiwa T: Pollen tube attraction by the synergid cell. Science 2001, 293:1480-1483. 25. Chen YH, Li HJ, Shi DQ, Yuan L, Liu J, Sreenivasan R, Baskar R, Grossniklaus U, Yang WC: The central cell plays a critical role in pollen tube guidance in Arabidopsis. Plant Cell 2007, 19:3563-3577. In contrast to previous findings, the authors of this paper demonstrate that in addition to the cells of the egg apparatus, the central cell also plays a role in pollen tube attraction. 26. Lausser A, Kliwer I, Srilunchang K, Dresselhaus T: Sporophytic control of pollen tube growth and guidance in maize. J Exp Bot 2010, 61:erp330. 27. Hu¨lskamp M, Schneitz K, Pruitt RE: Genetic evidence for a longrange activity that directs pollen tube guidance in Arabidopsis. Plant Cell 1995, 7:57-64. 28. Diboll AG, Larson DA: An electron microscopic study of the mature megagametophyte in Zea mays. Am J Bot 1966, 53:391-402.
44. Higashiyama T, Kuroiwa H, Kawano S, Kuroiwa T: Explosive discharge of pollen tube contents in Torenia fournieri. Plant Physiol 2000, 122:11-14. 45. Michard E, Alves F, Feijo JA: The role of ion fluxes in polarized cell growth and morphogenesis: the pollen tube as an experimental paradigm. Int J Dev Biol 2009, 53(6) doi: 10.1387/ ijdb.072296em. 46. Cordts S, Bantin J, Wittich PE, Kranz E, Lo¨rz H, Dresselhaus T: ZmES genes encode peptides with structural homology to defensins and are specifically expressed in the female gametophyte of maize. Plant J 2001, 25:103-114. 47. Mouhat S, Jouirou B, Mosbah A, De Waard M, Sabatier JM: Diversity of folds in animal toxins acting on ion channels. Biochem J 2004, 378:717-726.
29. Jensen WA: The ultrastructure and histochemistry of the synergids of cotton. Am J Bot 1965, 52:238-256.
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31. Ma´rton ML, Dresselhaus T: A comparison of early molecular fertilization mechanisms in animals and flowering plants. Sex Plant Reprod 2008, 21:37-52.
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51. Punwani JA, Rabiger DS, Lloyd A, Drews GN: The MYB98 subcircuit of the synergid gene regulatory network includes genes directly and indirectly regulated by MYB98. Plant J 2008, 55:406-414. 52. Silverstein KA, Moskal WA Jr, Wu HC, Underwood BA, Graham MA, Town CD, VandenBosch KA: Small cysteine-rich peptides resembling antimicrobial peptides have been underpredicted in plants. Plant J 2007, 51:262-280. 53. Graham MA, Silverstein KAT, VandenBosch KA: Defensin-like genes: genomic perspectives on a diverse superfamily in plants. Crop Sci 2008, 48:S1-S11. 54. Ramamoorthy V, Zhao X, Snyder AK, Xu JR, Shah DM: Two mitogen-activated protein kinase signalling cascades mediate basal resistance to antifungal plant defensins in Fusarium graminearum. Cell Microbiol 2007, 9:1491-1506.
This study together with [56] reports the first examples of plant defensins (here tomato DEF2) with a dual function in defense and developmental signaling. 57. Jackson S, Heath I: Roles of calcium-ions in hyphal tip growth. Microbiol Rev 1993, 57:367-382. 58. Ausubel F: Are innate immune signalling pathways in plants and animals conserved? Nat Immunol 2005, 6:973-979. 59. Lehrer RI: Multispecific myeloid defensins. Curr Opin Hematol 2007, 14:16-21. 60. Shiu SH, Karlowski WM, Pan R, Tzeng YH, Mayer KF, Li WH: Comparative analysis of the receptor-like kinase family in Arabidopsis and rice. Plant Cell 2004, 16:1220-1234. 61. Dolan L: Body building on land: morphological evolution of land plants. Curr Opin Plant Biol 2009, 12:4-8.
55. Allan A, Snyder A, Preuss M, Nielsen E, Shah D, Smith T: Plant defensins and virally encoded fungal toxin KP4 inhibit plant root growth. Planta 2008, 227:331-339. This study together with [57] reports the first examples of plant defensins with a dual function in defense and developmental signaling.
62. Sprunck S, Baumann U, Edwards K, Langridge P, Dresselhaus T: The transcript composition of egg cells changes significantly following fertilization in wheat (Triticum aestivum L.). Plant J 2005, 41:660-672.
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Current Opinion in Plant Biology 2009, 12:773–780
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Micropylar pollen tube guidance and burst: adapted from defense mechanisms? Thomas Dresselhaus and Mihaela L Ma´rton After the first description of fertilization in flowering plants some 125 years ago (Strasburger E: Neue-Untersuchungen u¨ber den Befruchtungsvorgang bei den Phanerogamen als Grundlage fu¨r eine Theorie der Zeugung. Gustav Fischer; 1884), we are finally beginning to understand the various molecular mechanisms leading to sperm delivery and discharge inside the hidden micropylar region of the female gametophyte (embryo sac). The last phase of pollen tube guidance culminating in tube burst and explosive release of tube contents requires extensive crosstalk between both male and female gametophytes. The first molecules identified that play key roles in these processes represent highly polymorphic proteins, similar to major components of the plant innate immune system. Here we summarize recent advances and briefly discuss the underlying molecular mechanisms also in respect to prezygotic barriers of reproductive isolation. Address Cell Biology and Plant Physiology, University of Regensburg, 93053 Regensburg, Germany Corresponding author: Dresselhaus, Thomas ([email protected])
Current Opinion in Plant Biology 2009, 12:773–780 This review comes from a themed issue on Cell Biology Edited by Jirˇı´ Friml and Karin Schumacher Available online 4th November 2009 1369-5266/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2009.09.015
Introduction During the evolution of flowering plants, their sperm cells have lost mobility and are delivered to the female gametes, egg, and central cell, via the vegetative pollen tube cell to achieve double fertilization. The journey of the two sperms cells via the pollen tube, from its germination site at the papillae cells of the stigma, through the transmitting tract inside the style toward the target female gametophyte (embryo sac) is assisted by intensive communication between the tube cell and surrounding sporophytic maternal tissues. The past two decades have seen tremendous progress in our understanding of the underlying molecular mechanisms of pollen tube germination and growth, sporophytic guidance as well as various self-incompatibility systems occurring between www.sciencedirect.com
haploid pollen/pollen tube and diploid cells of the pistil. Excellent reviews have been published [1–4] and these processes will not be further considered here. In contrast, until recently there was little progress in elucidating the molecular mechanisms of the femalegametophyte-controlled last phases of pollen tube growth and guidance, growth arrest, sperm discharge and transport as well as gamete fusion. This is due to the fact that the female gametophyte, also referred to as the ‘hidden generation’ [5], is deeply embedded in the maternal tissues of the ovary and ovule. However, the characterization of novel mutants [6,7,8,9,10], reverse genetic approaches [11,12,13,14], in vitro methods [12,15], and the development of various gamete-specific markers as well as techniques such as spinning disk live-time imaging [16,17] finally enabled the visualization of the cellular processes preceding plant gamete fusion and the establishment of appropriate first mechanistic models. We will therefore restrict our review on micropylar guidance, and inter-gametophytic signaling preceding pollen tube growth arrest and burst, and comment on the evolution of some key molecules involved that might contribute to prezygotic reproductive isolation barriers in plants [18–20].
Gametophytic pollen tube attraction After emerging from the transmitting tissue, the male gametophyte (pollen tube) is thought to be guided by chemotactic factors secreted by the synergid cells of the female gametophyte. This hypothesis is supported by genetic, cell ablation, and in vitro investigations, using excised Arabidopsis thaliana and Torenia fournieri ovules, showing that short-range pollen tube guidance is controlled by the female gametophyte via species-specific chemotactic signals [15,21–24]. Taking advantage of the naked embryo sac of T. fournieri, laser-assisted cell ablation experiments showed that the two synergid cells are necessary and sufficient to attract the pollen tube. Egg and central cells are either not involved in pollen tube attraction in T. fournieri, or not able to produce/secrete sufficient amounts of the guidance molecule. In maize, it seems that the egg cell contributes to the synthesis of the attraction signal [11], and in Arabidopsis even the central cell was suggested to play a critical role in pollen tube attraction [25]. However, it cannot be ruled out that the corresponding CCG (CENTRAL CELL GUIDANCE) gene encoding a potential transcriptional regulator acts rather indirect and is instead required, for example, for embryo sac maintenance. Current Opinion in Plant Biology 2009, 12:773–780
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The maximum distance of attraction in vitro is about 200 mm in T. fournieri [2], and about 100 mm in Arabidopsis [14,15] and maize (A Lausser et al. [26], Sporophytic control of pollen tube growth and guidance in maize, unpublished data), which contradicts models of longrange guidance by the female gametophyte [27]. As estimated mathematically, the maximum distance of gametophyte attraction is strongly limited by its production and not expected to significantly exceed this distance [22]. Mature synergid cells contain large numbers of secretory organelles such as stacked arrays of endoplasmic reticulum, Golgi complexes as well as vesicles in a secretory zone in proximity to the micropylar filiform apparatus [22,28–30]. This extracellular structure is generated by cell wall invaginations of both synergids and is composed mainly of cellulose, hemicellulose, and glycoproteins. It is thus likely that this apoplastic structure can take up and distribute vesicle contents secreted by the synergid cells. In conclusion, we suggest to consider the synergid cells as the ‘glandular cells’ of the female gametophyte similar to follicle cells in mammals [31]. Owing to the occurrence of high Ca2+ concentrations in the synergid cells, Ca2+ has long been considered a potential attractant [32]. But elevation of the Ca2+ ion concentration in in vitro pollination assays using the above described T. fournieri system did not affect the observed attraction [21], indicating that it does not serve as an attractant at all or is only part of an attractant cocktail of molecules. A gamma-amino butyric acid (GABA) gradient that is established along the pollen tube pathway was also suggested to guide the tube toward the micropylar region of the ovule [33]. However, in vitro pollen tube attraction studies using synthetic GABA have not been performed to demonstrate that it indeed functions as a pollen tube attractant. Moreover, various in vitro studies have indicated that pollen tube guidance and sperm release depends on species-specific factors rather than on general molecules such as Ca2+ or GABA. The first gene identified that encoded a candidate micropylar attractant of the pollen tube secreted by the female gametophyte was ZmEA1 (Zea mays EGG APPARATUS1), which is specifically expressed in the egg apparatus (egg and synergid cells) of maize and downregulated after fertilization. ZmEA1 encodes a polymorphic precursor protein of 94 amino acids and was shown to be secreted to the cell walls of micropylar nucellus cells of mature embryo sacs. Pollen tubes arrived at the micropyle of ZmEA1 knockdown plants without penetrating the intercellular space of micropylar nucellus cells providing further support that ZmEA1 might be involved in micropylar pollen tube guidance [11]. However, a biochemical proof was still lacking and we therefore show here (Figure 1a and b) that an N-terminal cleaved predicted mature ZmEA1 protein of 49 amino acids can directly attract maize pollen tubes in vitro at a concentration of <10 mM. Secreted cysteine-rich proteins (CRPs) named Current Opinion in Plant Biology 2009, 12:773–780
Figure 1
In vitro pollen tube attraction and burst studies using recombinant or chemically synthesized proteins. (a) 10 mM ZmEA1 was mixed with Alexa Fluor 488 and released in the proximity (ca. 30 mm) of an active maize pollen tube using a micro-capillary. (b) Around 22 min after application, pollen tube tip growth was reoriented toward the region of ZmEA1 droplet release (asterisk). (c) LURE1 (TfCRP1) and Alexa Fluor 488containing gelatin beads (asterisk) were placed about 50 mm from the growing pollen tube tip (arrowhead). Around 20 min after application, the pollen tube tip (double arrowheads) changed growth direction toward the LURE1-containing bead. (d) Addition of ZmES4 induced pollen tube burst at the very tube tip (arrow) and explosive discharge of its contents within <1 min. Scale bars: 50 mm.
LUREs have been recently identified as female gametophyte attractants in T. fournieri [12]. These polymorphic proteins are specifically synthesized in synergid cells and belong to a subgroup of the defensin-like (DEFL) super gene family of CRPs. Immunolocalization showed that LURE1 and LURE2 are secreted to the filiform apparatus. Recombinant mature proteins can attract competent pollen tubes at nanomolar concentrations in vitro in a species-specific manner (Figure 1c). Microinjection experiments of LURE morpholino antisense oligomers into mature embryo sacs significantly impaired pollen tube attraction providing further evidence that the DEFL genes LURE1 and LURE2 encode micropylar pollen tube attractants derived from the synergids. Results from the description of the GEX3 (GAMETE EXPRESSED3) gene of Arabidopsis for a plasma-membrane-located protein containing four PQQ (b propeller) domains [34] are difficult to interpret. Although GEX3 is most strongly expressed in pollen and according to promoter-marker expression at a low level in egg cells, reciprocal crossing experiments using transgenic lines indicate that the observed defect of micropylar pollen tube guidance is caused by the female gametophyte. Whether the observed phenotype is produced by an www.sciencedirect.com
Micropylar pollen tube guidance and burst Dresselhaus and Ma´rton 775
Figure 2
Hypothetical model showing the cellular and molecular mechanisms involved in micropylar pollen tube attraction and sperm discharge. (a) Vesicles containing pollen tube attractants are steadily secreted from mature synergid cells. After binding to pollen-tube-located receptor(s), pollen tube tip growth is reoriented (large arrowhead) in a concentration-dependent manner toward the synergid cells. While vesicles containing pollen tube ligands are also steadily discharged at the tube tip, secretory vesicles containing pollen tube ‘toxins’ are accumulating in the secretory zone of the synergids, but are not discharged. (b) Pollen tube invades the filiform apparatus (black region) between both synergid cells signaling toward the synergid cells via plasma-membrane-located proteins such as FER/SIR and LRE. Activated synergid cells discharge ‘toxin’-containing vesicles leading to pollen tube growth arrest via modulation/activation of pollen tube receptors/ion channels. NO and ROS play a yet unclear role during this process. (c) Intergametophytic signaling induces cell death in the receptive synergid cell and pollen tube burst after a dramatic change in the micro-environment of the pollen tube tip due to synergid degeneration and ion channel opening. Abbreviations: dSY, degenerated synergid cell; MGU, male germ unit consisting of two sperm cells and vegetative nucleus; PT, pollen tube; pSY, persistent synergid cell; SY, synergid cell; vNC, nucleus of the vegetative tube cell.
egg cell or embryo sac maturation defect is unclear, as egg cell identity was not analyzed. A number of reports have demonstrated that secretion of the attractant(s) and thus micropylar pollen tube guidance occurs only when embryo sacs are fully mature [15,22]. In magatama (maa) mutants of Arabidopsis, for example, an embryo sac maturation defect leads to pollen tube growth on the funicular surface, but not inside the micropyle [23,35] indicating both that first, pollen tube guidance until the last, micropylar phase, is sporophytically controlled and that second, only mature embryo sacs are capable of generating/secreting the micropylar attractant(s). Moreover, mutations in a synergid-expressed gene encoding the transcription factor MYB98 show an immature and less organized filiform apparatus; as a consequence, micropylar pollen tube guidance is affected [14]. It was also observed that after the first pollen tube has successfully released its contents, further attraction significantly decreases and second pollen tubes are very rarely found in the micropyle of wild type ovules of various plant species, despite the persistence of the second synergid cell [11,15,24]. This finding indicates that the attractant(s) are degraded, inactivated or no longer secreted, or that additional pollen tubes are actively repulsed. www.sciencedirect.com
Inter-gametophytic signaling preceding pollen tube growth arrest After arrival at the micropyle, the pollen tube directly enters one of the two synergid cells (the receptive synergid) via the filiform apparatus in plant species such as Arabidopsis or T. fournieri [14,22]. In grasses, the embryo sac is additionally embedded by a few layers of stack-like oriented nucellus cells, which have to be passed before the tube can enter the embryo sac [11,27]. After contact with the receptive synergid cell, the latter initially undergoes programmed cell death in Arabidopsis, maize, and many other plant species [36], followed by pollen tube rupture, which generally occurs within a few minutes [2,9], and discharge of its contents including the two sperm cells to achieve double fertilization (Figure 2). This poorly understood process is referred to as pollen tube reception and relies on extensive cell–cell communication between male and female gametophytes [37] via the pollen tube tip and the synergid cell(s) [38]. In recent years, a few Arabidopsis mutants have been described with impaired sperm cell release. In heterozygous feronia ( fer) [39] and sire`ne (sir) [40] mutants about half of the ovules remained unfertilized, although pollen tubes properly penetrated the micropylar region, but Current Opinion in Plant Biology 2009, 12:773–780
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partially failed to arrest its growth and instead continued to grow inside the embryo sac. Moreover, mutant pollen tubes failed to rupture and did not release sperm cells. Molecular cloning revealed that FER/SIR encodes the same plasma membrane localized receptor-like kinase (RLK) belonging to the CrRLK1L-1 subfamily consisting of 17 kinases [7]. Although FER/SIR is also expressed during vegetative development, its function in ovules is associated with the synergids where the protein seems to be asymmetrically distributed at the surface of the synergid cells accumulating in the plasma-membrane invaginations of the filiform apparatus [7]. It was therefore suggested that FER/SIR perceives unknown signals derived from the pollen tube tip to induce a signaling cascade to arrest pollen tube growth. In addition to the identification of the pollen tube derived ligand(s), it still remains to be shown whether FER/SIR signaling induces the release of a feedback signal to the pollen tube, is involved in proper synergid maturation required to release factors that control pollen tube growth, or induces synergid cell death [38] as a prerequisite of pollen tube growth arrest and burst. The identification and molecular characterization of the Arabidopsis LORELEI (LRE) gene provides further support for the existence of gametophyte signaling or recognition mechanisms preceding pollen tube burst [8]. The LRE mutant, resembling the fer/sir phenotype, encodes a small plant specific CRP probably attached to the plasma membrane via a glucosylphosphatidylinositol (GPI)-anchor. Although the authors did not show subcellular localization of LRE at the synergid surface, the gene is specifically expressed in synergids of mature ovules suggesting that it might accumulate at its cell surface and is involved in signaling or recognition mechanisms required to arrest further pollen tube growth. An unusual mutant impaired in pollen tube reception is amc (abstinence by mutual consent). The fer/sir pollen tube overgrowth phenotype occurs only when amc pollen tubes reach amc female gametophytes [10]. AMC encodes a peroxin with limited homology to PEX13 involved in peroxisomal protein import. Peroxisomal targeting signaldependent protein import into pollen peroxisomes is completely disrupted in an amc null mutation. A number of small general molecules such as reactive oxygen species (ROS) and nitric oxide (NO) are produced in peroxisomes [41] and NO has recently been shown to reorient pollen tube growth in a negative chemotropic manner. Additionally, a role of NO in modulating Ca2+ signaling was proposed [42,43]. Whether NO and other small molecules such as ROS modulate the activity of other factors or are directly required for the gametophyte dialogue to reorient or inhibit further tube growth, to induce synergid cell death or induce sperm release remains to be shown by further experimentation. Current Opinion in Plant Biology 2009, 12:773–780
Pollen tube burst Live time observations of pollen tube burst using the T. fournieri in vitro system showed that the pollen tube tip is located within the filiform apparatus between two intact synergid cells before discharge. It was further shown that the plasma membrane of the receptive synergid ruptures within a second at its tip and discharge is explosive [44]. However, the molecular mechanisms leading to pollen tube burst are unclear. As discussed above, Ca2+ has long been considered a key molecule in the fertilization process in plants. Without doubt Ca2+ plays an important role, for pollen tube tip growth [45] and high concentrations have also been determined to exist in the synergid cells [22,32]. It is thus not a surprise that a pollen tube plasmamembrane Ca2+ pump ACA9 (autoinhibited Ca2+ ATPase 9) is required for normal pollen tube growth and sperm discharge. Mutant aca9 pollen tubes reach the micropyle and embryo sac, cease growth, but fail to burst and release their contents [9]. This finding suggests that pollen tube growth arrest and tube burst are separate processes and that sperm discharge requires Ca2+ signaling. DEFL genes ZmES1-4 specifically expressed in the mature maize embryo sac that are immediately downregulated after fertilization [46] have now been shown to be able to directly induce pollen tube burst within <1 min in vitro (S Amien et al., Defensin-like ZmES4 mediates pollen tube burst in maize via opening of the potassium channel KZM1, unpublished data. This is the first report of a molecular mechanism leading to pollen tube burst. DEFL protein ZmES4 induces pollen tube burst in a species-specific manner in maize. Pollen tube membrane depolarization occurs in vitro after ZmES4 application and is accompanied by opening of the inward rectifying K+ channel KZM1) (Figure 1d). Knockdown of ZmES genes leads to normal micropylar pollen tube attraction, but pollen tube contents are not discharged. In vitro pollen tube growth assays showed that mature ZmES4 leads to pollen tube rupture in a species-specific manner accompanied by membrane depolarization preceding burst. The K+ channel KZM1 was identified as a direct target of ZmES4. Whether ZmES proteins additionally act at other targets such as Ca2+ channels, for example the maize ortholog of Arabidopsis ACA9, similar to animal toxins that can modulate various channels simultaneously [47], and thus induces Ca2+ signaling (see above), should be clarified in future experimentations. As shown in Figure 2 it is not unlikely that conserved, but species-specific FER/SIR, LRE, or Ca2+ inter-gametophyte signaling mechanisms are involved in the release of ZmES4 in maize and orthologous proteincontaining vesicles in other plant species. Before fertilization ZmES4 is visible in the secretory zone of the synergids (Figure 2) and vesicles might be released upon signals received from the invading pollen tube, mechanistically similar to the release of cortical granule contents from fertilized animal egg cells/oocytes [31]. www.sciencedirect.com
Micropylar pollen tube guidance and burst Dresselhaus and Ma´rton 777
Pathogen defense and gametophyte communication involve similar molecular players Remarkably, ZmES4 as well as LURE1 and LURE2 belong to various subclasses of the same super gene family encoding DEFL-proteins in plants [48,49]. Mature defensins (DEFs) and DEFLs are an ancient class of small cysteine-rich antimicrobial proteins/peptides that play an important role in the innate immune system of plants and invertebrates. Plant DEFs and DEFLs have been identified and purified from several tissues, but transcripts are especially abundant in the reproductive tissues of the flower and mature seed. Many DEFL genes are expressed in female gametophyte cells [12,13,46,50,51]. Genome surveys identified >300 DEFL genes in Arabidopsis and around 130 genes in rice [52]. The majority of genes are clustered in the genome and seem to have evolved by duplication and positive selection [53]. However, until recently plant DEFs/DEFLs were mainly shown to be involved in defense where they act as protein translation inhibitors, a-amylase inhibitors, microbial inhibitors, zinc tolerance mediators, ion channel blockers, or protease inhibitors [49]. Most characterized plant DEFs have antifungal activity that inhibits hyphal elongation, with up to 100% growth inhibition and partial lysis of susceptible fungi [48]. However, the exact mode of action of plant DEFs is not yet entirely unraveled. Some plant DEFs with antifungal inhibitory seem to activate an intracellular signaling pathway (e.g. a mitogen-activated protein kinase, or MAPK, signaling pathway) that changes the structural integrity of fungal membranes and culminates in membrane permeabilization [48,54]. This in turn may result in increased Ca2+ influx, K+ efflux, and reduced fungal growth. Moreover, such intracellular signaling cascades correlate with the induction of ROS and NO generation. ROS excess is known to trigger apoptotic cell death in yeast [48] and might also play a role in synergid cell death. It is possible that signaling molecule/s secreted by the cells of the female gametophyte modulate/s intracellular signaling cascades in pollen tubes to generate an excess of ROS via activated peroxisomes leading almost simultaneously to synergid cell death and pollen tube burst. However, since the above-mentioned AMC gene encoding a peroxin was identified in both plant male and female gametophytes [10], it might also be the other way round, that the pollen tube secretes signaling molecule/s modulating intracellular processes including ROS production in the receptive synergid cell, thus triggering cell death. Beyond their role in defense against microbial infection, additional/novel functions of DEFs/DEFLs were acquired in plant development and reproduction. The first examples shown were that they can inhibit the growth of roots and root hairs in A. thaliana [55] as well as pollen viability and seed production [56]. In addition, www.sciencedirect.com
modulation of DEF2 expression in tomato pleiotropically altered the growth of various organs and interfered with fungal invasion by inhibiting tip growth of Botrytis cinerea. Similar to pollen tube and root hair tip growth, fungal hyphal tip growth is a process controlled by a gradient in cytosolic Ca2+ generated by hyphal tip-localized Ca2+ channels [57]. It was thus suggested that DEF2 may alter Ca2+ transport [56] by blocking Ca2+ channels. In animals, most toxins related to plant DEFs/DEFLs were shown to modulate K+ channels, Na+ channels, or Ca2+-activated K+ channels either as pore blockers or as gating modifiers [47]. Whether ZmES4 involved in pollen tube burst solely acts on K+ channels or additionally on others such as Ca2+ channels, whether the target(s) of LURE1 and LURE2 are ion channels or RLKs, and the identification of the molecular mechanisms of NO and ROS action in gametophyte communication are exciting questions for future research.
Evolutionary aspects of defense and reproduction in plants All living organisms have developed efficient mechanisms to defend themselves against pathogen attack. Plants and animals use various intracellular and membrane receptors for the detection of pathogen-associated molecular patterns, serine/threonine kinases for downstream processing, and MAPKs for signal relay. Similar defense responses are elicited, resulting in the generation of NO and ROS, the production of antimicrobial compounds including DEFs, finally culminating in the hypersensitive response or programmed cell death [48,58]. Owing to the lack of an adaptive immune system, it is likely that ancient plants interacted with a broad range of microbial pathogens with different lifestyles and infection strategies, and dedicated a relatively large portion of their genome to defend against pathogens [53]. DEF genes, for example, have been found in all eukaryotic kingdoms and were proposed to have a common ancestor that arose more than a billion years ago [59]. Additionally, the number of plant plasma-membrane RLKs is multiplied compared with animals [60]. In conclusion, the evolutionary arms race between plants and their attackers probably provided ancient water-plants already with a highly sophisticated pool of polymorphic ligand and receptor proteins involved in defense, hundreds of millions of years before going onto land around 460 million years ago [61]. It is thus likely that this pool of genes, which has evolved through positive selection, was also used as a molecular basis to establish novel species-specific gametophytic communication mechanisms required in land plants. Further support for this hypothesis is provided, for example, by the analysis of the dual role of DEF2 in tomato [56] as well as ZmES4 and FER/SIR activity: ZmES4-overexpressing Arabidopsis plants, for example, recover more quickly from fungal attack and, in contrast to wild type plants, fungal hyphae growth is absent a few weeks after infection (S Amien et al., Defensin-like ES4 Current Opinion in Plant Biology 2009, 12:773–780
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mediated pollen tube rupture in maize accompanies opening of the potassium channel KZM1, unpublished data). Moreover, homozygous fer/sir mutants are resistant to fungal infection (U Grossniklaus and co-workers, unpublished data) indicating first, that these proteins are functional and possess targets both in defense and in reproduction and second, that members of these large families evolved de novo function(s) adapted from defense mechanisms according to novel environmental requirements after going onto land.
The research community has finally established the necessary tools and methods to study these hidden processes and to elucidate the underlying molecular mechanisms and their evolution.
Acknowledgements We are grateful to Tetsuya Higashiyama and Irina Kempel for providing images of LURE1 pollen tube guidance and ZmES4 pollen tube burst, respectively. The German Research Foundation (DFG, grant MA 3976/1-1) is acknowledged for financial support to MLM.
References and recommended reading Conclusions The identification of the first molecular players involved in micropylar pollen tube guidance and burst shed new insights into the evolution and functional diversification of polymorphic proteins involved in plant reproduction. It is noteworthy that the angiosperm female gametophyte expresses many more CRP subgroups [12,13,46,50,51,62] as well as members of other polymorphic gene families [7,11] whose functions are completely unknown. Functional studies remain challenging and are complicated by the fact that most of the genes described above occur as gene families showing strongly overlapping expression patterns. The majority of proteins described above seem to have been adapted from defense mechanisms and their species-specific activity may represent a component of reproductive isolation, essential to speciation [19,20]: interspecific pollination experiments within various plant genera resulted, for example, in the above described fer/sir phenotype. Pollen tube overgrowth in the embryo sac was observed after interspecific crosses of Rhododendron [63], as well as with crosses of various Brassicaceae pollen on Arabidopsis stigmata [7]. When ovules of Scrophulariaceae species related to T. fournieri were cultivated together, pollen tubes were significantly more attracted to synergid cells of the corresponding species [21]. These findings indicate that, similar to micropylar pollen tube guidance and burst, inter-gametophytic communication to arrest pollen tube growth is dependent on speciesspecific signaling mechanisms. These findings therefore provide further molecular support that the mechanisms and driving forces of prezygotic barriers to genetic exchange are stronger than postzygotic barriers. Pollen-expressed genes like the polymorphic DEFL-similar sterility-locus cysteine-rich (SCR/SP11) genes and their stigma-expressed receptor kinases of Brassica rapa, as well as various F-box proteins, have been considered as incompatibility factors involved in sporophytic self-incompatibility as one prezygotic barrier [1]. We discussed various incompatibility factors involved in female gametophytic control of sperm delivery, adding first, micropylar pollen tube guidance; second, pollen tube growth arrest; and third, its burst as further prezygotic barriers. In conclusion, it has never been more exciting to study fertilization mechanisms in plants. Current Opinion in Plant Biology 2009, 12:773–780
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