Recent work has revealed signaling molecules that control pollination, including small peptides that mediate pollen recognition and glycoproteins that support pollen tube growth. The polarized growth of pollen tubes requires a calcium-mediated signal cascade, and cues derived from the haploid and diploid ovule cells guide pollen tubes to the eggs.
Addresses
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA
*e-mail: [email protected]
Current Opinion in Plant Biology1999, 2:18–22 http://biomednet.com/elecref/1369526600200018 © Elsevier Science Ltd ISSN 1369-5266
Abbreviations
AGP arabinogalactan protein
PCP pollen coat protein
SI self-incompatibility
SLG S-locus glycoprotein
SRK S-locus receptor kinase
TTS transmitting tissue-specific glycoprotein
Introduction
Plants, like other organisms, invest enormous resources to find the most suitable mate. Although regulation of flow-ering time and insect pollination help ensure that pollen arrives at an appropriate flower, recent studies reveal the importance of communication between male and female cells in controlling plant mating. Cell signaling regulates recognition of pollen by the stigma, migration of pollen tubes through the pistil, delivery of sperm to the ovules, and finally, co-ordinated development of the zygote, endosperm, seed, and fruit (Figure 1). In this review, we summarize recent insights into the mechanisms that con-trol pollination and fertilization.
Pollen recognition at the stigma surface — the
first line of defense
Stigma cells use a variety of mechanisms to limit the suc-cess of inappropriate pollen. On dry stigmas, the transfer of water and nutrients to pollen grains is also controlled — foreign pollen often remains dehydrated. The invasion of pollen tubes into stigma cells is regulated in many species, including those with wet stigmas. These early checkpoints serve to block pollination before pollen consumes valuable resources within the pistil.
In self-incompatible Brassica species, self fertilization is inhibited at the stigma surface. A genetic and molecular characterization of self-incompatibility (SI) defined a com-plex polymorphic locus, which contains both male and female-specific genes [1•]. Recent studies conclusively
demonstrate that two stigma-specific genes are necessary
for SI: SLG encodes an extracellular glycoprotein and SRK encodes a receptor kinase localized to the plasma mem-brane [1•,2]. Mutations in either gene result in
self-compatibility, as do transgenic constructs that reduce their expression levels [1•,3,4].
How do SLG and SRK enable plants to reject incompati-ble pollen? Current models propose an interaction with a pollen-specific S locus gene, initiating a phosphorylation cascade that results in pollen rejection. Although the SI response is often accompanied by a rapid and localized production of β-1,3 glucan, or callose, in the stigma, sur-prising recent studies demonstrate that callose is not required for SI [5]. In addition, though adhesion of pollen to the stigma is species specific [6], pollen binding affinity does not correlate with SI [7•].
One approach used to identify pollen-specific SI compo-nents relied on characterizing the structure of the Slocus itself. This resulted in the identification of one promising gene, SLA (S-locus anther), that is disrupted by a retro-transposon insertion in several self-compatible mutants [8]. Linkage of this SLA sequence defect with a mutant phenotype initially led to proposals that SLAmediates SI in pollen [8]. Subsequent investigations, however, showed that many wild-type strains have a similar insertion within SLA, indicating that SLA is not necessary for a functional SI response [9]. Nonetheless, exploring the S-locus for other genes active in SI remains a valuable approach.
Purification of pollen components that bind to SLG and SRK led to the exciting discovery that peptide signals may regulate SI [10••]. The extracellular pollen coating
con-tains several proteins, including a large family of cysteine-rich peptides (pollen coat proteins or PCPs) released onto the stigma surface upon pollination [10••,11,12•]. Purified fractions containing these peptides
bind to SLG, although their function remains undefined. Curiously, the PCP genes neither map to the S locus, nor do their products bind to SLG in an allele-specific manner. Because genetic evidence suggests the male determinant of SI ought to be polymorphic and within the S-locus, a search for additional SLG-binding components may prove fruitful. Nonetheless, the strong sequence similarity between the PCPsand defensins, plant polypeptides with antifungal activity [12•], presents an intriguing possibility
for peptide signaling during pollination.
Immediately after pollination, compatible pollen grains absorb water and begin to form a pollen tube that invades the stigma. Whether stigma surfaces are wet or dry, lipids have been implicated in mediating pollen hydration [13,14,15•]. Mutations that eliminate the lipid-rich
exu-date of wet Nicotiana stigmas result in female sterility, and
addition of lipids to the surface of these stigmas restores fertility [15•,16]. In plants with dry stigmas, long-chain
lipids in the pollen coating are required for hydration [13,14]; delivery of those lipids may require oleosins — oil binding proteins that solubilize lipid droplets [17–19]. Though the application of short-chain lipids can cause pollen hydration even on dry stigmas [15•], it remains to be
demonstrated if lipids alone can trigger a normal series of hydration events. Lipids may serve to form a water-tight seal between pollen and the stigma, facilitating the rapid transport of water through channels in the stigma and pollen membranes. The recent identification of an aqua-porin-like gene within the Brassica S-locus may provide clues toward the regulation of this process [20]. Self-com-patible mutants show reduced expression of this aquaporin, though the nature of the mutant defect remains to be demonstrated. Finally, it is reasonable to expect that several components contribute to pollen germination, some such as lipids or water channels that are shared among all plants and others, like flavonols, that play a species-specific role [21,22].
Pollen tube growth —organizing cell polarity
Once pollen tubes invade the stigma, they grow through the style at rates that can approach 1 cm/hr. This polarized cell growth has been the focus of recent investigations:revealing parallels with systems as diverse as root hairs, yeast buds, and neural outgrowths. Small GTP-binding proteins are required for polarized secretion in many organisms; similarly, Rho GTPases are localized at the tips of pollen tubes and are essential for tube growth [23,24]. Rapid pollen tube growth also requires remodeling of pis-til cell walls, and expansin and extensin-like activities required for cell wall loosening have been purified from maize pollen tubes [25,26].
Although polarized secretion and cell wall loosening are clearly required for pollen tube growth, additional activi-ties are likely to provide signals that orientate the growing tubes. Calcium has long been implicated in directing tube growth, and exciting new evidence reveals the pathways and mechanisms by which pollen tubes react to calcium gradients [27,28]. Intriguingly, calcium gradients within pollen tubes oscillate in phase with pulses of tube growth [29,30•]. Identifying the source, concentration, and
destination of the calcium ions that pulse across the pollen tube tip may provide important clues as to how calcium regulates cell polarity. Pathways known to be regulated by calcium are also under investigation, and a calcium-depen-dent protein kinase has been implicated in pollen tube reorientation [31]. Growing pollen tube tips exhibit high levels of a calcium-dependent, calmodulin-independent
Figure 1
Anatomy of fertilization in flowering plants.
(a)Representation of a typical pistil, consisting of a stigma, style, and ovary. Pollen grains land on the stigma surface and germinate pollen tubes which grow into the style through a central transmitting tract which contains the ovules. (b)Desiccated pollen grains land on the stigma cell surface; once compatible grains are hydrated, they are able to produce a pollen tube that carry two sperm to each ovule. sc, stigma cell; dp, desiccated pollen grain; hp, hydrated pollen grain; pt, pollen tube. (c)
After exiting the style, pollen tubes enter the ovary, where they approach individual ovules. Each ovule contains a haploid female gametophyte, consisting of the egg and central cell nuclei (cn), that is surrounded by diploid integument tissue (it). The tubes grow up the funiculus and enter the micropyle, an opening in the ovule that allows the tube access to the female gametophyte. Fertilization of the egg by one sperm cell forms the zygote, and fusion of a second sperm cell with the two central cell nuclei forms the endosperm. fu, funiculus; pt, pollen tube.
Stigma
Style Growing pollen tube
Ovary
pt
it cn
egg hp
dp
fu
sc
pt
(a) (b)
(c)
Transmitting tract
Pollen grain
protein kinase (CDPK); localization of kinase activity changes with pollen tube orientation. Further, release of caged calcium ions on one side of the pollen tube apex induces a localized increase in calcium concentration, tube reorientation, and a corresponding increase in kinase activ-ity adjacent to the site of release [28,31].
Pollen tube guidance — from the style to
the ovules
As pollen tubes travel through the style, many interac-tions between male and female cells facilitate inhibition of incompatible pollen tubes. In Nicotiana species, self-incompatible pollen tubes are arrested by S locus RNases [32,33]. These RNase molecules vary with each S allele, and current models suggest their import into or activity within incompatible pollen is regulated by a male-specif-ic S-encoded protein. An intense search for this putative pollen component is underway. Unfortunately, analysis of the structure and function of the S-RNase genes them-selves reveal few clues. Though active site residues have been identified [32], construction of chimeric RNases from different S alleles failed to identify functional domains [34].
Compatible pollen tubes import nutrients from the style extracellular matrix and respond to guidance signals as they travel through the transmitting tract. Arabinogalactan proteins (AGPs), abundant in stylar secretions, may pro-vide important directional cues. The Yariv reagent, which binds AGPs, inhibits growth of lily and maize pollen, although a similar effect was not observed in all plants [35]. In Nicotiana tabacum, a transmitting tissue specific glyco-protein, TTS, exhibits a gradient of glycosylation that may attract and stimulate growth of pollen tubes to the base of the pistil [36,37]. Similar results, however, were not obtained with Nicotiana alata; despite 97% sequence iden-tity and similar sugar modifications, the TTS homolog GaRSGP fails to exhibit a glycosylation gradient, attract pollen tubes or stimulate tube growth [38]. These recent studies warrant careful consideration and substantial work will be required to identify conserved style components that regulate the growth of pollen tubes.
After exiting the style, pollen tubes grow toward the ovules, diploid structures that contain haploid female gametophytes (Figure 1). Though it was previously unclear which ovule tissues contribute to pollen tube guid-ance, recent studies show that both diploid and haploid female cells are important. Several diploid-specific muta-tions have been identified that alter ovule structure and function (see K Schneitz review, this issue pp 13–17, and [39•]). Some of these dramatically affect ovule
morpholo-gy; not surprisingly, pollen tube guidance is also aberrant [40]. Other diploid-specific alterations reveal that morpho-logically normal ovules can also be defective in guiding pollen tubes [40,41]. In one case, female tissues from a self-sterile Arabidopsismutant exhibit aberrant pollen tube guidance only in the presence of mutant pollen [41]. This
suggests diploid pistil cells, including those that comprise the funiculus and integuments (Figure 1), may be required to attract, bind, and promote the growth of pollen tubes.
Conclusive evidence that the haploid gametophyte is also required for pollen tube targeting has come from the identi-fication of an Arabidopsis strain that contains a balanced chromosomal translocation [42••]. Heterozygotes that carry
this translocation have a normal genetic complement; in these plants, half of the meioses undergo adjacent chromo-some segregation, resulting in lethal chromochromo-some imbalances in the meiotic products. Consequently, although all of the ovules have genetically normal diploid cells, half contain aborted gametophytes. In these plants, pollen tubes fail to approach the abnormal gametophytes, though normal ovules have associated pollen tubes. Although these studies reveal that a female gametophyte is important for guidance, the female gametophyte might induce the surrounding diploid tissue to emit guidance cues.
The next generation — from gametophytes
to seeds
Gene expression in pollen tubes and female gametophytes is required for successful reproduction, but few genetic screens aimed at identifying haploid-specific (gametophyt-ic) mutants have been performed. Diploid-specific (sporophytic) mutations dramatically reduce fertility and are relatively easy to isolate; in contrast, male or female-specific gametophytic defects cause at most a 50% reduction in seed yield. Recently, progress has been made toward identifying large numbers of defects in Arabidopsis gametophytes. One approach has relied on following the transmission of a heterozygous T-DNA insertion to the next generation; biased transmission indicates a defect in the fertility of the male and/or female gametophytes that carry the insertion [43,44]. Alternatively, genes with inter-esting haploid-specific roles in development can be identified by careful characterization of mutants with defective pollen morphology [45,46].
The genomes contributed by the male and female game-tophytes may be fundamentally different, due to heritable epigenetic modifications (genetic imprinting) that alter gene expression patterns. In a recent study, crosses between Arabidopsis diploids, tetraploids, and hexaploids were performed to cause imbalances in the maternal and paternal contributions to the embryo and endosperm [47•]. An excess of maternal genomes (for
development in Arabidopsishave been identified through gene trapping, a transposon insertion screen that results in the fusion of a reporter gene carried on the transposon to chromosomal genes [50]. Mutations in MEDEA, a homolog of Drosophila Polycomb genes, cause embryo abortion when transmitted through females. Though the number of genes that can be imprinted is unknown, genetic strategies aimed at isolating maternally or pater-nally imprinted loci will likely prove worthwhile.
The co-ordinated formation of embryos, endosperm, seeds, and fruit is likely to require the exchange of mul-tiple developmental signals, and mutations that affect this signaling have been identified. Arabidopsis mutants known as fie (fertilisation-independent endosperm) or fis ( fer-tilisation-independent seed) [51•,52], inappropriately initiate
development of the endosperm before fertilization takes place. Mutant fie and fis alleles often fail to be transmit-ted through female gametophytes. Even when fertilization of these aberrant gametophytes takes place, the defect in developmental co-ordination results in embryo lethality. Regulation of fruit development likely requires a distinct set of genes; mutation of a MADS-box gene (AGL8 or FRUITFULL) was recently shown to interrupt fruit development, without affecting the forma-tion of seeds [53].
Conclusion
Plant reproduction occurs in a competitive environment — choosing among the available mating partners requires an amazing array of cell signaling interactions. Recent investiga-tions of pollination in self-compatible and self-incompatible plants have unearthed a complex set of genetic and molecu-lar controls, active in both diploid and haploid cells. Though many important molecules have been defined, discerning those that play general, rather than species-specific, roles remains a challenge for the future.
References and recommended reading
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