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The pollen grain and the pollen tube that grows from it are complex entities which must respond to a diverse array of signals to carry out their roles in sexual reproduction. Research is beginning to reveal the nature both of the signals and of the signal transduction machinery that converts these signals into directional, polarized growth.
Addresses
Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA;
e-mail: [email protected]
Current Opinion in Plant Biology1999, 2:419–422
1369-5266/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved.
Introduction
Plant life-cycles, unlike those found in animals, alternate the growth of the diploid sporophytic organism with the growth of another form, the haploid gametophyte. In more primitive vascular plants such as ferns, the gametophyte grows as a free living organism that is responsible for pro-ducing the gametes. The gametes then undergo fertilization to produce the next generation of recognizable sporophytic ferns. The growth and development of the gametophyte in these organisms is obviously complex and includes such interesting processes as sex determination, which is under both genetic and hormonal control [1]. In the angiosperms the growth and development of the game-tophytes is greatly reduced, although the programs of development are still complex and still contain many processes which depend on sophisticated control through cell–cell signaling and genetic regulatory circuits. In fact, from the point of view of a developmental geneticist, the growth and development of the angiosperm male gameto-phyte between pollination and fertilization is a system full of fascinating events, with virtually every aspect of male development dependent on and/or under the control of the female reproductive system.
This review will describe a number of these reproductive interactions, beginning with the arrival of the pollen grain at the stigmatic surface where it must adhere, hydrate and germinate — all processes which can be regulated by male/female interactions in at least some species. Following germination, the growing pollen tube must suc-cessfully navigate the interior of the female reproductive system, a process that requires correct perception of female guidance cues and that can be arrested by some forms of self-incompatibility. Finally, after the pollen tube has delivered sperm cells to the embryo sac, the sperm cells themselves must interact with their targets (the egg and the central cell) in order for double fertilization to take place.
In the beginning: control of germination
When the mature male gametophyte leaves the anther it has typically undergone a dramatic desiccation process. The desiccated pollen grain is dispersed via one of several mechanisms and, with luck, eventually finds itself residing on a receptive stigma. An important factor in determining the success of the pollen grain in landing on a stigma is the ability of the pollen grain to adhere tightly to the receptive surface. This adhesion has been studied in some detail in Brassica, where it has been shown to depend on the glyco-proteins SLG and SLR1 located in the stigmatic cell wall [2•]. Interestingly, even though SLG is a product of the Brassica S-locus and varies with S-haplotype, adhesion does not seem to be dependent on S-haplotype in Brassica [3], nor does pollen–stigma adhesion seem to be species specific in the crucifer family [4]. Pollen from species out-side the crucifer family do show reduced affinity for crucifer stigmas, however [4].
Once the pollen grain has managed to adhere to a stigmat-ic surface it must rehydrate prior to germinating and producing a pollen tube. In species with dry stigmas there is no readily available source of water for rehydration and this constitutes an important barrier for the male gameto-phyte to overcome. The barrier to hydration serves as an important control point in sporophytic self-incompatibility systems [5], interspecific incompatibility systems [6] and in a more general family-specific type of recognition seen in Arabidopsis[7]. In Arabidopsis, pollen grains from any mem-ber of the crucifer family hydrate freely on the surface of the stigma [7], a pattern similar to that seen for adhesion of pollen to the stigma surface [2•]. Lack of a more specific recognition system in Arabidopsis may not be surprising considering the apparent loss from the Arabidopsisgenome of the entire polygenic S-locus responsible for self-incom-patibility in Brassica [8••]. Genetic analysis has revealed that this family level recognition depends on the presence of long chain lipid components found in the tryphine layer on the outside of the pollen grain [7,9], a finding which is consistent with recent results implicating lipids in pollen germination and directional growth [10••,11]. Despite large scale genetic screens that have been performed in Arabidopsisto identify mutations disrupting pollen–stigma interactions, an additional new locus with this mutant phe-notype has been identified by knocking out a gene encoding an enzyme related to fatty acid elongase (FAE) [12]. As the ‘mutants’ at this new locus were generated by sense suppression of the endogenous gene, it is possible that the phenotype observed is due to simultaneous down-regulation of several genes with related DNA sequences, explaining why this locus was not identified using conventional genetic approaches. Further implication of the role of lipids in the pollen–stigma interaction comes from the the observation that changes in the lipid biochemistry of
the epidermal cuticle of vegetative cells can lead to their ability to interact with pollen grains in a manner which mim-ics the stigmatic surface [13]. Recent results have identified mutations in several genes which produce this phenotype [14], leading to the suggestion that the lipids found in the tryphine layer and the cuticle on the epidermal surface may play a very direct role in the regulation of water transfer between stigmatic cell and pollen grain [15].
How do we get there from here: pollen tube
growth and guidance
Once a pollen grain has hydrated on the surface of the stig-ma, it must germinate and grow a pollen tube. This tube must grow into the tissues of the female reproductive sys-tem to gain access to the interior of the ovary. Inside the ovary, individual pollen tubes will be guided to individual ovules where they will discharge their sperm cells as a pre-requisite to fertilization. An effective combination of molecular and cell biology has led to the identification of a number of molecular components involved in the growth of pollen tubes, and a number of these may be involved in pollen tube guidance as well.
A gradient of Ca2+ions found at the tip of growing pollen tubes has long been implicated in their growth [16]. Recent experiments have demonstrated that fluxes in intracellular Ca2+concentrations are correlated with peak growth pulses [17,18], whereas cytoplasmic acidification and current influx may be involved in termination of the growth pulses [19•]. Blockade of Ca2+channels with La3+ or Gd3+leads to the loss of pulsating growth, implicating at least some Ca2+channels in this process [20], and localiza-tion of calmodulin to specific sites at the tip of the pollen tube also appears to be essential for tip growth [21]. Experiments have also demonstrated that Ca2+plays a role in control of oriented pollen tube growth [22], possibly through a Ca2+-dependent protein kinase [23•]. Molecular experiments have also identified a number of receptor pro-tein kinases specific to pollen tubes, although a function for these has not yet been demonstrated [24,25]. Rho-relat-ed GTPases may also have an important role to play in tip growing pollen tubes [26].
Pollen tube growth through the style is also a develop-mental control point, this time for gametophytic self-incompatibility systems. Two types of systems have been characterized: one involving S-Rnases, which are expressed in the transmitting tissue of the style in many solanaceous species [27]; and another, found in poppies, which involves low molecular weight proteins of unknown biochemical function [28]. In both cases the gene products that have been characterized act on the female side of the interaction, whereas their male partners are unknown. In the poppy system it has been possible to develop an in vitroassay system involving pollen tube arrest when grown in the presence of either style extracts or recombinant S -gene products [29]. This in vitro system has allowed a serious structure/function analysis of S-proteins to be
performed and has led to the identification of specific amino acid residues that are required for pollen tube growth inhibition and might also allow the identification of those residues responsible for allelic specificity [30••].
To reach its target — the ovule — the growing pollen tube must respond to guidance cues in the female reproductive system. The nature of the signals involved in this guidance process are largely unknown, although different groups have provided evidence for both long range chemotactic signals [31,32] and short range, adhesion mediated signals [33]. A possible candidate for a short range signal was iden-tified as a transmitting tract specific glycoprotein [34,35], but more recent work has raised doubts about the validity of earlier conclusions [36]. Work in identifying the signals involved has long been hampered by the lack of a good in vitro system for demonstrating pollen tube guidance to ovules. A first step towards such a system has recently been achieved in Torenia [37••]. Interestingly, pollen tube guidance in vitro was shown to be dependent on pollen germination on the stigma, implying that activation of the guidance system requires pollen–stigma interaction. Variation in the nature of the signals may be responsible for the different pathways followed by pollen tubes in differ-ent species — whereas most pollen tubes differ-enter the ovule via the micropyle, in some species the tube enters the nucellus at the chalazal end via the funiculus [38].
What do we do when we get there:
double fertilization
On arrival at the ovule, the pollen tube of angiosperms delivers two sperm cells, both of which are required for a successful double fertilization event. Typically, one of the sperm cells is closely associated with the vegetative nucle-us of the pollen tube during pollen tube growth, whereas the other is said to be ‘unassociated’. In the case of Arabidopsis sperm, the cells complete the S phase of the cell cycle as the pollen tube grows through the gynoecium, completing replication just in time for double fertilization to occur [39•]. During this time other changes appear to take place in the sperm cells as well — sperm isolated early in the growth process have a marked tendency to fuse with one another and this tendency diminishes as the pollen tube elongates [40]. One of the sperm cells fertilizes the egg cell to produce the zygote (and ultimately the embryo), whereas the other fertilizes the central cell to produce the endosperm. This type of double fertilization, which leads to a seed containing a single embryo plus endosperm, is a unique adaptation of angiosperms and is presumably derived from a system similar to that found in the Gnetales where the two fertilization events lead to two zygotes [41]. In many species of angiosperms the distinc-tion between the two fertilizadistinc-tion events is even further refined. In these species the two sperm cells are morpho-logically distinguishable (due to variation in organellar content, for example) and the two sperm cells can be shown to preferentially fertilize either the egg or the cen-tral cell [42]. Thus, not only are the products of double
fertilization different in the angiosperms, but the sperm cells giving rise to the two products are different as well.
What distinguishes these two sperm cells? Are their differ-ences strictly due to segregation of cytoplasmic contents when the generative cell divides or do they each express a unique set of genes as well, which contributes to their unique cellular identities? Do these differences also exist in species where the two sperm cells are morphologically indistinguishable? What signals and receptors are responsi-ble for the preferential fertilization of the egg or central cell? Several recent experiments have brought us to the point where we can begin to examine these exciting ques-tions. First, the production of cDNA libraries by PCR-based methods has allowed libraries to be made from very small quantities of isolated tissue. These methods have been exploited to construct libraries representing genes expressed specifically in the generative cell, the immediate cellular precursor to the sperm cells [43•]. In addition to the identification of a generative cell-specific gene of unknown function, these libraries have also led to the identification of generative cell-specific histone genes [44]. By taking advantage of the known morphological dif-ferences between the two sperm of Plumbago zeylanica it has been possible to isolate the two types as discrete pop-ulations [45•]. Surely it is only a matter of time before we are able to identify genes which are specifically expressed in each of the two sperm cell types?
Conclusions
Both classic and current genetic experiments have defined a number of key signals which regulate the germination, growth and development of the pollen grain and tube. Molecular and cellular tools are now defining the bio-chemical nature of the machinery that responds to those signals. In the near future we can look forward to a time when these two approaches will be successfully integrated to allow a reasonably complete characterization of the mol-ecular mechanisms which govern sexual reproduction in higher plants.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest ••of outstanding interest
1. Eberle JR, Banks JA: Genetic interactions among sex-determining genes in the fern Ceratopteris richardii.Genetics
1996, 142:973-985.
2. Luu D-T, Marty-Mazars D, Trick M, Dumas C, Heizmann P:
• Pollen–stigma adhesion in Brassicaspp involves SLG and SLR1 glycoproteins.Plant Cell1999, 11:251-262.
This paper, together with [3,4], describes a careful set of experiments which use hydrostatic floatation to measure the adhesion between pollen grain and stigma. Using transgenic lines to suppress expression of specific genes, or antibodies directed against specific proteins, it has been possible to directly measure the roles played by specific molecules in the adhesion interaction.
3. Luu D-T, Heizmann P, Dumas C: Pollen–stigma adhesion in kale is not dependent on the self-(in)compatibility genotype.Plant Physiol1997, 115:1221-1230.
4. Luu D-T, Passelegue E, Dumas C, Heizmann P: Pollen–stigma capture is not species discriminant within the Brassicaceae
family. Comptes-Rendus-de-l’Academie-des-Sciences-Serie-III-Sciences-de-la-Vie1998, 321:747-755.
5. Sarker RH, Elleman CJ, Dickinson HG: Control of pollen hydration in Brassicarequires continued protein synthesis, and
glycosylation is necessary for intraspecific incompatibility.Proc Natl Acad Sci USA1988, 85:4340-4344.
6. Hiscock SJ, Dickinson HG: Unilateral incompatibility within the
Brassicaceae: further evidence for the involvement of the self-incompatibility (S)-locus.Theor App Gen1993, 86:744-753. 7. Hülskamp M, Kopczak SD, Horejsi TF, Kihl BK, Pruitt RE:
Identification of genes required for pollen–stigma recognition in
Arabidopsis thaliana.Plant J1995, 8:703-714.
8. Conner JA, Conner P, Nasrallah ME, Nasrallah JB: Comparative
•• mapping of the Brassica Slocus region and its homeolog in
Arabidopsis: implications for the evolution of mating systems in the Brassicaceae.Plant Cell1998, 10:801-812.
This study describes the characterization of a large region of the Brassica
and Arabidopsisgenomes surrounding the site of the S-locus in Brassica. Although these regions show a high degree of synteny, none of the S-locus specific sequences appear to be present in Arabidopsis, providing a simple explanation for its lack of self-incompatibility. As the S-locus may also play a role in interspecific incompatibility, the absence of these sequences may explain broad acceptance by Arabidopsisof pollen from any species that is a member of the crucifer family.
9. Preuss D, Lemieux B, Yen G, Davis RW: A conditional sterile mutation eliminates surface components from Arabidopsispollen and disrupts cell signaling during fertilization.Genes Dev1993,
7:974-985.
10. Wolters-Arts M, Lush WM, Mariani C: Lipids are required for
•• directional pollen-tube growth.Nature1998, 392:818-821. Experiments in this paper define classes of lipids that can substitute for stig-matic exudate in Nicotianato allow pollen hydration and germination. These lipids will also allow pollen tubes to penetrate the epidermal surfaces of leaves. Together with experiments described in [11], the authors demon-strate a role for these lipids in the initial polarized growth of the pollen tube.
11. Lush WM, Grieser F, Wolters-Arts M: Directional guidance of
Nicotiana alata pollen tubes in vitro and on the stigma.Plant Physiol1998, 118:733-741.
12. Millar AA, Clemens S, Zachgo S, Giblin EM, Taylor DC, Kunst L: CUT1, an Arabidopsis gene required for cuticular wax
biosynthesis and pollen fertility, encodes a very-long-chain fatty acid condensing enzyme.Plant Cell1999, 11:825-838.
13. Lolle SJ, Berlyn GP, Engstrom EM, Krolikowski KA, Reiter W-D, Pruitt RE: Developmental regulation of cell interactions in the
Arabidopsis fiddlehead-1mutant: a role for the epidermal cell wall and cuticle.Dev Biol1997, 189:311-321.
14. Lolle SJ, Hsu W, Pruitt RE: Genetic analysis of organ fusion in
Arabidopsis thaliana.Genetics1998, 149:607-619.
15. Lolle SJ, Pruitt RE: Epidermal cell interactions: a case for local talk. Trends Plant Sci1999, 4:14-20.
16. Pierson ES, Miller DD, Callaham DA, Shipley AM, Rivers BA, Cresti M, Hepler PK: Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media.Plant Cell1994,
6:1815-1828.
17. Messerli M, Robinson KR: Tip localized Ca2+pulses are coincident
with peak pulsatile growth rates in pollen tubes of Lilium longiflorum.J Cell Sci1997, 110:1269-1278.
18. Holdaway-Clarke TL, Feijo JA, Hackett GR, Kunkel JG, Hepler PK:
Pollen tube growth and the intracellular cytosolic calcium gradient oscillate in phase while extracellular calcium influx is delayed.Plant Cell1997, 9:1999-2010.
19. Messerli M-A, Robinson K-R: Cytoplasmic acidification and current
• influx follow growth pulses of Lilium longiflorumpollen tubes. Plant J1998, 16:87-91.
This paper demonstrates the presence of pulses of both H+and current influx
in pollen tubes undergoing pulsatile growth. Unlike changes in the Ca2+
con-centration that are correlated directly with growth pulses, the H+and current
pulses lag peak growth rate by several seconds leading the authors to pro-pose that they may be involved in the termination of growth pulses.
20. Geitmann A, Cresti M: Ca2+channels control the rapid expansions
in pulsating growth of Petunia hybridapollen tubes.J Plant Phys
1998, 152:439-447.
21. Moutinho A, Love J, Trewavas AJ, Malhó R: Distribution of calmodulin protein and mRNA in growing pollen tubes.Sex Plant Reprod1998, 11:131-139.
22. Malhó R, Trewavas AJ: Localized apical increases of cytosolic free calcium control pollen tube orientation.Plant Cell1996,
8:1935-1949.
23. Moutinho A, Trewavas AJ, and Malhó R: Relocation of a Ca2+
• dependent protein kinase activity during pollen tube reorientation. Plant Cell1998, 10:1499-1509.
Experiments in this paper measure the in vivoactivity of a protein kinase in pollen tubes. In growing pollen tubes the activity is tip localized, but shows a more uniform distribution in static tubes. Manipulations that cause reorien-tation of the growing tip also alter the localization of the kinase activity and do so in a manner that is predictive of the new growth orientation.
24. Mu J-H, Lee H-S, Kao T-H: Characterization of a pollen-expressed receptor-like kinase gene of Petunia inflataand the activity of its encoded kinase.Plant Cell1994, 6:709-721.
25. Muschietti J, Eyal Y, McCormick S: Pollen tube localization implies a role in pollen-pistil interactions for the tomato receptor-like protein kinases LePRK1 and LePRK2.Plant Cell1998, 10:319-330. 26. Li H, Wu G, Ware D, Davis KR, Yang Z: ArabidopsisRho-related
GTPases: differential gene expression in pollen and polar localization in fission yeast.Plant Physiol1998, 118:407-417. 27. Lee H-S, Huang S, Kao T-H: S proteins control rejection of
incompatible pollen in Petunia inflata.Nature1994, 367:560-563. 28. Franklin-Tong VE, Ride JP, Read ND, Trewavas AJ, Franklin FCH: The self-incompatibility response in Papaver rhoeasis mediated by cytosolic free calcium.Plant J1993, 4:163-177.
29. Franklin-Tong VE, Ride JP, Franklin FCH: Recombinant stigmatic self-incompatibility (S-) protein elicits a Ca2+transient in pollen of Papaver rhoeas.Plant J1995, 8:299-307.
30. Kakeda K, Jordan ND, Conner A, Ride JP, Franklin-Tong VE, Franklin •• FCH: Identification of residues in a hydrophilic loop of the
Papaver rhoeas Sprotein that play a crucial role in recognition of incompatible pollen.Plant Cell1998, 10:1723-1732.
The experiments described in this paper take advantage of the elegant in vitro
incompatibility system which is available in Papaver. Site-directed mutants in the Sprotein are constructed and their ability to inhibit pollen tubes of various genotypes assayed. The results define regions of the protein which are essen-tial for activity and are a big step towards defining the basis of allelic speci-ficity, an extremely important result in any recognition system.
31. Hülskamp M, Schneitz K, Pruitt RE: Genetic evidence for a long-range activity that directs pollen tube guidance in Arabidopsis. Plant Cell1995, 7:57-64.
32. Ray SM, Park SS, Ray A: Pollen tube guidance by the female gametophyte.Development1997, 124:2489-2498.
33. Wilhelmi LK, Preuss D: Self-sterility in Arabidopsisdue to defective pollen tube guidance.Science1996, 274:1535-1537.
34. Cheung AY, Wang H, Wu HM: A floral transmitting tissue-specific glycoprotein attracts pollen tubes and stimulates their growth. Cell1995, 82:383-393.
35. Wu HM, Wang H, Cheung AY: A pollen tube growth stimulatory glycoprotein is deglycosylated by pollen tubes and displays a glycosylation gradient in the flower.Cell1995, 82:395-403.
36. Sommer-Knudsen J, Lush WM, Bacic A, Clarke AE: Re-evaluation of the role of a transmitting tract-specific glycoprotein on pollen tube growth.Plant J1998, 13:529-535.
37. Higashiyama T, Kuroiwa H, Kawano S, Kuroiwa T: Guidance in vitro •• of the pollen tube to the naked embryo sac of Torenia fournieri.
Plant Cell1998, 10:2019-2032.
Studies on the guidance of pollen tubes have long been hampered by the lack of a good in vitro system in which the tubes are guided specifically to their natural targets. The authors of this manuscript have at least partially overcome this problem, demonstrating a system in which at least a fraction of the tubes are guided. More importantly, they demonstrate that the guid-ance observed is dependent on live ovules as targets and on the germina-tion of the pollen grains on the stigma. These are two important results in their own right and also validate the specificity of the guidance observed.
38. Martinez PE, Herrero M: Pollen tube pathway in chalazogamous
Pistacia veraL.Int J Plant Sci1998, 159:566-574. 39. Friedman WE: Expression of the cell cycle in sperm of
• Arabidopsis: implications for understanding patterns of gametogenesis and fertilization in plants and other eukaryotes. Development1999, 126:1065-1075.
A nice and carefully performed study demonstrating that sperm cells in
Arabidopsisare actively engaged in DNA synthesis as they travel down the pollen tube. This result not only has important practical and evolutionary implications which are discussed by the author, but also clearly demon-strates that sperm cells are active cellular participants in fertilization rather than passive partners.
40. Tian HQ, Russell SD: The fusion of sperm cells and the function of male germ unit (MGU) of tobacco (Nicotiana tabacumL.).Sex Plant Reprod1998, 11:171-176.
41. Friedman WE: The evolution of double fertilization and endosperm: an ‘historical’ perspective.Sex Plant Reprod1998,
11:6-16.
42. Southworth D, Strout G, Russell SD: Freeze-fracture of sperm of
Plumbago zeylanicaL. in pollen and in vitro.Sex Plant Reprod
1997, 10:217-226.
43. Xu H, Swoboda I, Bhalla PL, Singh MB: Male gametic cell-specific
• gene expression in flowering plants.Proc Natl Acad Sci USA
1999, 96:2554-2558.
PCR was used to construct a cDNA library from isolated lily generative cells. Random screening of the library allowed identification of a clone which was specifically expressed in the generative cell, LGC1. In situ hybridization experiments demonstrated that LGC1 was expressed after generative cell formation and continued to be expressed in the sperm cells following gen-erative cell division.
44. Xu H, Swoboda I, Bhalla PL, Singh MB: Male gametic cell-specific expression of H2A and H3 histone genes.Plant Mol Biol1999,
39:607-614.
45. Zhang Z, Xu H, Singh MB, Russell SD: Isolation and collection of
• two populations of viable sperm cells from the pollen of
Plumbago zeylanica.Zygote1998, 6:295-298.
