LUREs

Chapter 6

LUREs Tetsuya Higashiyama

ABSTRACT The pollen tube is a tip-growing cell that emerges from pollen grains. It contains two immotile sperm cells and transports them to the ovule, which contains the egg cell. Pollen-tube guidance to the ovule is critical for the successful fertilization of flowering plants. The existence of chemotropic factors derived from the ovule was postulated >140 years ago in the late 1860s. Ovular attractants, called LURE peptides, were first identified in 2009 in the model plant Torenia fournieri. LUREs are defensin-like peptides that are specifically and abundantly expressed in two synergid cells adjacent to the egg cell. LUREs show severe species preference and attraction specificity and might be involved in reproductive isolation. Unlike SCR/SP11, another defensin-like peptide for self-incompatibility in flowering plants, many LUREs are probably involved in generating the attraction signal for the pollen tube.

DISCOVERY The study of pollen-tube guidance and associated attractants has a long history.4,7 Chemotropic attractant factors derived from the ovule of flowering plants were first postulated as early as the 1860s22 when researchers observed that pollen tubes grow toward the ovule in vitro. Since then, many plant biologists have attempted but failed to identify attractants derived from the ovule. These classical biochemical experiments were based on various bioassay systems and led to the identification of external calcium ions as a possible attractant in snapdragons.18 However, external calcium ions are necessary for the growth of the tip of the pollen tube.8 Attraction and growth stimulation were not clearly distinguished in these systems.7,8 One difficulty associated with bioassays of pollen-tube attraction is that pollen tubes still grow, but in random directions, without specific attractants if the medium contains the requisite compounds to sustain tube growth, such as water, calcium, borate, and sucrose.8 In animals, the chemoattractant protein for the neuronal growth cone, netrin, was identified in 1994 using an in vitro assay.14 This study showed that neural cells emerge from tissues only when netrin proteins are present in the medium. Handbook of Biologically Active Peptides. http://dx.doi.org/10.1016/B978-0-12-385095-9.00006-3 Copyright © 2013 Elsevier Inc. All rights reserved.

In 1998, an in vitro system was developed in Torenia fournieri, a unique plant with a protruding female gametophyte.10 The haploid female gametophyte, known as the embryo sac, usually has one egg cell and two synergid cells at the micropylar end, three antipodal cells at the opposite chalazal end, and one huge central cell in the middle.5 Pollen tubes were directly attracted to the protruding micropylar end of this female gametophyte (Figure 1; for movies7). This was consistent with genetic analyses in Arabidopsis thaliana showing that the female gametophyte governed pollen-tube guidance to the ovule.11 Laser ablation experiments showed that the attraction signal in T. fournieri was derived from the two synergid cells on the side of the egg.12 This attraction activity has severe species preference and specificity even in closely related species,9 suggesting that it is probably a rapidly evolving molecule(s). Thus, genes expressed in synergid cells of Torenia were examined using manually collected synergid cells to identify attractant candidates among the peptides and proteins.20 The synergid cell showed a unique gene expression profile: various cysteine-rich peptides (CRPs) are abundantly expressed. Three major CRPs, TfCRP1, TfCRP2, and TfCRP3, were analyzed, and recombinant peptides of TfCRP1 and TfCRP3 showed strong activity to attract pollen tubes. These peptides were named LUREs (TfCRP1 as LURE1 and TfCRP3 as LURE2). Knockdown of LURE1 or LURE2 genes by injecting morpholino antisense oligomers impaired pollen-tube attraction. These results indicate that LURE1 and LURE2 are diffusible attractant molecules derived from the synergid cell of the ovule (Figure 2).20 Genomic PCR analysis of TcCRP1 from a closely related species, Torenia concolor, identified a LURE1 homolog that also showed species-preferential attraction activity (Figure 2).13

STRUCTURE OF THE PEPTIDE The apparent molecular masses of mature LURE1 and LURE2 are 8.6 and 9.8 kD, respectively, based on Western blotting of endogenous LUREs from the pistil.20 Cleavage sites of the signal peptide of LURE1 and LURE2 have been precisely predicted using the SignalP program. 31

Chapter | 6  LUREs

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The molecular weights of these endogenous LUREs are similar to those of recombinant LURE peptides expressed in Escherichia coli (predicted mature peptides lacking N-terminal signal sequences). These recombinant peptides of LURE1 and LURE2 show pollen-tube attraction activity at very low concentrations, as described below. Thus, it has been proposed that LURE1 and LURE2 are cleaved as predicted but are not substantially processed further. Secretion of endogenous LURE1 and LURE2 was confirmed by immunostaining, as described below.20 The predicted mature peptides of LURE1 and LURE2 contain six cysteines. The molecular structures of the LUREs have not been resolved, but correct intramolecular disulfide bonds between the six cysteines have been suggested to be critical for their activity. Refolding is critical for pollen-tube attraction by recombinant LURE peptides, and refolded LUREs are mainly monomers.20

DISTRIBUTION OF THE mRNA/PEPTIDE LURE1 and LURE2 genes are specifically expressed in the two synergid cells on the side of the egg cell, independent of pollination, as shown by reverse transcription polymerase chain reaction (RT-PCR) of isolated female cells

FIGURE 1  Pollen-tube attraction in the in vitro Torenia system. Two pollen tubes are attracted to the protruding embryo sac of Torenia fournieri. Pollen tubes are touching the filiform apparatus of two synergid cells in the embryo sac. Sequential images are shown and time after the start of observation is indicated as mm:ss. Modified after Ref. 7. Scale bar = 30 µm.

and immunostaining of ovules.20 LURE1 and LURE2 peptides are secreted directionally toward the micropylar end of the egg and accumulate in the filiform apparatus. The filiform apparatus is a structure formed by the ingrown and/or thickened cell walls of the two synergid cells, and might be involved in active secretion and/or protection of the female gametophyte from pollen-tube thrust.5 Accumulation of LURE peptides in the filiform apparatus is consistent with the behavior of pollen tubes; pollen tubes enter the female gametophyte through the region of the filiform apparatus.

BIOLOGICAL FUNCTIONS Pollen-tube guidance involves multistep controls exerted by various female tissues along the path of the pollen tubes.7 LURE peptides govern the last guidance step over short distances, possibly at 100–200 µm from the female gametophyte.21 The last guidance step is precise and is known as micropylar guidance.21 The existence of attractants in other guidance steps remains unclear. Chemocyanin, a basic cell wall protein expressed in the lily stigma, is a good guidance molecule candidate.15 Chemocyanin has ability to attract (reorient) pollen tubes in vitro; however, knockdown analyses have not been successful. It remains unknown whether LURE peptides are common in other flowering plants. Because CRPs generally show rapid molecular evolution, conventional homology searches are difficult for identifying LURE homologs in other plants. However, in Arabidopsis thaliana, CRPs are likely to be involved in synergid cell functions, including micropylar pollen-tube guidance.21 ZmEA1, a nondefensin-like peptide, has been suggested as a possible pollen-tube attractant candidate for micropylar guidance in Zea mays.2,17 An in vitro attraction assay using recombinant LURE peptides has been used for characterization (Figure 3).20 For quantitative analysis, recombinant peptides are embedded in gelatin beads approximately 40 µm in diameter and placed in front of each pollen tube using a micromanipulator to examine whether they attract pollen tubes. Pollen-tube attraction is a concentration-dependent process. At optimum concentrations, >60% of pollen tubes are attracted, which is similar to the frequency of attraction in manipulated ovules.20 Surprisingly, the minimum concentration is 40 pM for both LURE1 and LURE2,3,20 which means that only 1000 LURE molecules in the bead are sufficient to attract pollen tubes. Attraction of the pollen tube by LUREs might not simply

FIGURE 2  Alignments of LURE amino acid sequences. Alignments of LURE1, LURE2, and TcCRP1 (LURE1 homolog of Torenia concolor) are shown.13,20 TcCRP1 preferentially attracted pollen tubes of its own species.20 Conserved amino acids in more than two peptides are shaded gray. Conserved cysteines and glycines of the γ-core motif are shaded black and displayed in open boxes below the alignment.

SECTION | I  Handbook of Biologically Active Peptides: Plant Peptides

result from a LURE concentration gradient, as implied by visualization of LURE2 with Alexa labeling.3 The attraction activity of LUREs is similar to that of the synergid cell.20 T. fournieri pollen tubes are preferentially and specifically attracted to ovules of their own species in the presence of ovules of Torenia concolor or Lindernia micrantha.9 Consistently, pollen tubes of T. concolor and L. micrantha are less attracted and scarcely attracted, respectively, by LURE peptides from T. fournieri.13,20 In the in vitro Torenia system, pollen tubes are grown through a cut style, as tubes become competent by growing through the pistil. The pollen tubes cultivated in semi-in vitro conditions, but not tubes germinated in vitro, are attracted to the synergid cell. Consistently, only competent pollen tubes growing through a cut style are attracted by LURE ­peptides.20 When LUREs were initially identified, only three major CRPs expressed in the synergid cell were examined. However, there are even more CRPs expressed in synergid cells, and some of them are likely to be candidate LUREs.6 A mixture of plural or multiple LURE peptides might generate the attraction signal for micropylar guidance. It has also been proposed that LURE1 and LURE2 might have independent functions.6

RECEPTORS AND SIGNALING CASCADES Receptors for LUREs have not been identified. The severe species preferentiality and specificity of LURE activity imply that some partner molecules on the plasma membrane of the pollen tube, possibly with highly variable domains, bind to the LUREs. Because the direction of tip growth is controlled at the tip by changing the exit site of secretory vesicles, it is plausible that the receptor is localized at the 00:00

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FIGURE 3  Pollen-tube attraction by recombinant LUREs. Pollen tubes were attracted using a micropipette (left) or a gelatin bead (right). Recombinant LURE2 (left) and LURE1 (right) were used. Note that the pollen tubes changed their direction to orient toward the injected LURE2 (left top, an asterisk) and a gelatin bead containing LURE1 (right, an asterisk). In the right panel, an arrowhead marks the position of the tip of the pollen tube when a gelatin bead was placed, and double arrowheads indicate the tip of the pollen tube growing toward the bead. Sequential images are shown in the left panels, and time after applying peptides is indicated as mm:ss. Modified after Ref. 20. Scale bar =20 µm.

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tip of the pollen tube.21 Endocytosis actively occurs in the subapical region of the pollen tube for recycling of molecules at the tip.1 Thus, the receptor might also be recycled at the tip. Pollen-tube competence, as described above, might involve some modifications of the receptor and/or signaling cascades. Amino acid sequences are significantly divergent between LURE1 and LURE2, except for the conserved cysteines (Fig. 2).20 The difference in amino acid sequence between LURE1 of T. fournieri and its homolog, T. concolor (TcCRP1), is only eight amino acids; however, this difference leads to significant species specificity.13 Thus, because LURE1 and LURE2 are more highly diverged, they may bind to different receptors. Receptors for many plant peptide ligands, including defensin-like SCR/SP11, are serine–threonine receptor kinases with one transmembrane domain. However, the response of the pollen tube to LURE peptides is rapid and spatially precise. Involvement of other types of receptors has also been discussed.21 LURE-specific signaling cascades are unknown, but the directional signals from expected receptors are likely to modify signaling cascades related to tip growth of the pollen tube.21 Alterations in the distribution of tip-focused concentration gradients of internal calcium ions and cAMP change the direction of growth, as shown using caged compounds and inhibitors.16,19

REFERENCES 1. Cheung AY, Hm Wu. Structural and signaling networks for the polar cell growth machinery in pollen tubes. Annu Rev Plant Biol 2008;59:547–72. 2. Dresselhaus T, Márton ML. Micropylar pollen tube guidance and burst: adapted from defense mechanisms? Curr Opin Plant Biol 2009;12:773–80. 3. Goto H, Okuda S, Mizukami A, Mori H, Sasaki N, Kurihara D, et al. Chemical visualization of an attractant peptide, LURE. Plant Cell Physiol 2011;52:49–58. 4. Heslop-Harrison J. Pollen germination and pollen-tube growth. Int Rev Cytol 1987;107:1–78. 5. Higashiyama T. The synergid cell: attractor and acceptor of the pollen tube for double fertilization. J Plant Res 2002;115:149–60. 6. Higashiyama T. Peptide signaling in pollen–pistil interactions. Plant Cell Physiol 2010;51:177–89. 7. Higashiyama T, Hamamura Y. Gametophytic pollen tube guidance. Sex Plant Reprod 2008;21:17–26. 8. Higashiyama T, Inatsugi R. Comparative analysis of biological models used in the study of pollen tube growth. Plant Cell Monogr 2006;3:265–86. 9. Higashiyama T, Inatsugi R, Sakamoto S, Sasaki N, Mori T, Kuroiwa H, et al. Species preferentiality of the pollen tube attractant derived from the synergid cell of Torenia fournieri. Plant Physiol 2006;142:481–91. 10. Higashiyama T, Kuroiwa H, Kawano S, Kuroiwa T. Guidance in vitro of the pollen tube to the naked embryo sac of Torenia fournieri. Plant Cell 1998;10:2019–31.

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11. Higashiyama T, Kuroiwa H, Kuroiwa T. Pollen-tube guidance: beacons from the female gametophyte. Curr Opin Plant Biol 2003;6:36–41. 12. Higashiyama T, Yabe S, Sasaki N, Nishimura Y, Miyagishima S, Kuroiwa H, et al. Pollen tube attraction by the synergid cell. Science 2001;293:1480–3. 13. Kanaoka MM, Kawano N, Matsubara Y, Susaki D, Okuda S, Sasaki N, et  al. Identification and characterization of TcCRP1, a pollen tube attractant from Torenia concolor. Ann Bot 2011;108:739–47. 14. Kennedy TE, Serafini T, de la Torre JR, Tessier-Lavigne M. Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell 1994;78:425–35. 15. Kim S, Mollet J-C, Dong J, Zhang K, Park S-Y, Lord EM. Chemocyanin, a small basic protein from the lily stigma, induces pollen tube chemotropism. Proc Natl Acad Sci USA 2003;100:16,125–30. 16. Malho R, Trewavas AJ. Localized apical increases of cytosolic free calcium control pollen tube orientation. Plant Cell 1996;8:1935–49.

Chapter | 6  LUREs

17. Márton ML, Cordts S, Broadhvest J, Dresselhaus T. Micropylar pollen tube guidance by EGG APPARATUS 1 of maize. Science 2005;307:573–6. 18. Mascarenhas JP, Machlis L. Chemotropic response of Antirrhinum majus pollen to calcium. Nature 1962;196:292–3. 19. Moutinho A, Hussey PJ, Trewavas AJ, Malhó R. cAMP acts as a second messenger in pollen tube growth and reorientation. Proc Natl Acad Sci USA 2001;98:10481–6. 20. Okuda S, Tsutsui H, Shiina K, Sprunck S, Takeuchi H, Yui R, et al. Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature 2009;458:357–61. 21. Takeuchi H, Higashiyama T. Attraction of tip-growing pollen tubes by the female gametophyte. Curr Opin Plant Biol 2011;14:614–21. 22. Van Tieghem P. Végétation libre du pollen et de l’ ovule. Ann Sci Nat Bot 1869;12:312–28.