340 Biochemical Society Transactions (2014) Volume 42, part 2

Interactions between pollen tube and pistilcontrol pollen tube identity and sperm releasein the Arabidopsis female gametophyteAlexander R. Leydon*, Adisorn Chaibang*1 and Mark A. Johnson*2

*Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912, U.S.A.

AbstractFlowering plants have immotile sperm that develop within the pollen cytoplasm and are delivered to femalegametes by a pollen tube, a highly polarized extension of the pollen cell. In many flowering plant species,including seed crop plants, hundreds of pollen tubes grow towards a limited number of ovules. This systemshould ensure maximal fertilization of ovules and seed production; however, we know very little about howsignalling between the critical cells is integrated to orchestrate delivery of two functional sperm to eachovule. Recent studies suggest that the pollen tube changes its gene-expression programme in response togrowth through pistil tissue and that this differentiation process is critical for pollen tube attraction by thefemale gametophyte and for release of sperm. Interestingly, these two signalling systems, called pollen tubeguidance and pollen tube reception, are also species-preferential. The present review focuses on Arabidopsispollen tube differentiation within the pistil and addresses the idea that pollen tube differentiation definespollen tube identity and recognition by female cells. We review recent identification of genes that maycontrol pollen tube–female gametophyte recognition and discuss how these may be involved in blockinginterspecific hybridization.

IntroductionThe pollinated pistil is a system that integrates hundredsof individual cell–cell interactions to achieve maximal seedproduction. Consider the journey of a single pollen tube: itinteracts with several female cell types as it germinates on thestigma, enters the transmitting tissue of the style, turns out onto the ovary surface, grows up a funiculus, enters an ovule mi-cropyle, contacts a synergid cell, and bursts to release its cargoof two sperm [1,2] (Figures 1 and 2A). The two sperm cellsthen interact with the degenerated synergid cell before fusingwith the female gametes (the egg and central cell) to producean embryo and endosperm within a developing seed [3].

The complexity of the pollen–pistil system becomesapparent when you consider a model such as Arabidopsisthaliana that contains 50–60 ovules and hundreds of pollentubes (Figure 1). It is likely that each of the cell–cellinteractions listed above (and others) is mediated by multiplepollen tube and female cell proteins and other molecules(e.g. carbohydrates). It is also becoming clear that thesesignalling systems are not discrete or linear, but feed backon each other to optimize reproductive success. For example,it was shown recently that the fusion of sperm cells withfemale gametes prevents attraction of more pollen tubes toan already fertilized ovule [4–6]. Integration of gamete fusionand pollen-tube-attraction systems ensures that only a single

Key words: Arabidopsis, double fertilization, MYB transcription factor, pollen tube reception.

Abbreviations: ACA9, autoinhibited Ca2 + -ATPase 9; ANX, ANXUR; FER, FERONIA; LRE, LORELEI;

NTA, NORTIA; SIV, semi-in vitro.1Present address: Energy Biosciences Institute, University of California Berkeley, Berkeley,

CA 94720, U.S.A.2To whom correspondence should be addressed (email mark_johnson_1@brown.edu).

pair of sperm cells is delivered to the female gametophyteand that gamete fusion occurs successfully before an ovulebecomes incapable of attracting another pollen tube. Thesefindings suggest the interesting possibility that steps in theflowering plant reproductive process that were thought tobe distinct from each other are integrated in ways thatsignificantly enhance reproductive success.

Pollen tube reception is the cell–cell signalling systemthat results in cessation of pollen tube growth in thefemale gametophyte, synergid degeneration and pollen tuberupture/sperm release. Substantial progress has been made inA. thaliana towards identifying female components of thissignalling system (reviewed in [7]); however, relatively littleis known about how the female gametophyte senses pollentube arrival or about how the pollen tube prepares for ruptureand sperm release. In the present article, we review recentstudies that suggest that the pistil induces a specific pollentube gene-expression programme that is critical for pollentube reception. We discuss this as an example of how distinctcell–cell interaction systems are integrated to maximizereproductive success and how differentiation of the pollentube within the pistil may be essential for species-preferentialrecognition of the pollen tube by the female gametophyte.

Pollen tubes differentiate in response togrowth through the pistilPollen tube physiology changes when it grows throughfloral tissue. For example, pollen tubes must grow throughpistil explants to be able to respond to attractants in vitro[8–10]. These experiments use a SIV (semi-in vitro)

C©The Authors Journal compilation C©2014 Biochemical Society Biochem. Soc. Trans. (2014) 42, 340–345; doi:10.1042/BST20130223Bio

chem

ical

So

ciet

y T

ran

sact

ion

s

ww

w.b

ioch

emso

ctra

ns.

org

Regulation of Fertilization and Early Seed Development 341

Figure 1 Pollen tube subgroup 18 (S18) MYB transcription factors

respond to growth through the pistil

A schematic diagram of an A. thaliana ovary with 12 ovules (instead

of the actual 50–60). Pollen grains (red) are germinating pollen tubes

on the stigma. Pollen tubes target the female gametophyte (grey).

Mature pollen grains [represented with two sperm (black) and a nucleus

(grey)] express MYB101, and lower levels of both MYB97 and MYB120.

Transcription of MYB targets is low in, or absent from, pollen grains.

Growth through the stigma and style activate transcription of MYB97

and MYB120; the three MYBs activate target genes in the pollen tube.

The pollen tube factors that sense the pistil environment and activate

MYB expression are not yet known.

pollen-tube-guidance assay in which pollen tubes growthrough the stigma and a portion of style before growingon to the surface of pollen growth media towards ovules[10]. In Torenia, where LURE pollen tube attractants werefirst described [11], the ability to respond to attractantsin vitro increases with prolonged growth through pistil tissue[12]. However, when LURE-binding sites were assessed byincubating pollen tubes with LUREs followed by detectionwith anti-LURE antibodies, it was observed that, whereasgrowth in the pistil was required to generate LURE-bindingsites, prolonged exposure to the pistil did not increase thedetection of binding sites further [12]. These data suggestthat the pollen tube senses the pistil environment, that LUREreceptor (not yet identified) expression reaches a maximum

as a consequence, but that prolonged exposure to the pistilenvironment may activate the receptor.

Interestingly, a pair of A. thaliana pollen-tube-expressedmembrane-associated receptor-like cytoplasmic kinases(LIP1 and LIP2) were recently shown to be induced bygrowth in the pistil and required for the full response topurified LURE proteins in the SIV assay and for pollentube guidance in the pistil [13]. It is not yet clear whetherLIP1 and LIP2 are involved directly in LURE perception;however, these findings underscore the idea that pollentubes differentiate during growth in the pistil, resulting inexpression of proteins required to respond to pollen tubeguidance cues.

Pollen tube differentiation for spermrelease is controlled by three MYBsOver 1000 genes were detected in the transcriptome of pollentubes grown in the SIV assay that were not detected in pollentubes grown in vitro [14]. To identify regulators that controlpollen tube gene expression in response to the pistil, 26 SIV-induced transcription factors were identified [14]. This listincluded three closely related R2R3-MYB type transcriptionfactors [subgroup 18: MYB97, MYB101 and MYB120] [14].Single and double myb mutants did not display seed set de-fects; however, triple mutants had a ∼70% reduction in seedproduction [15]. myb triple mutant pollen grains and tubesdevelop normally, grow through the pistil and target ovulesnearly as efficiently as wild-type. These data suggest thatMYB97, MYB101 and MYB120 do not regulate expressionof the LURE receptors. However, upon interaction with thefemale gametophyte, myb triple mutants fail to arrest theirgrowth and ∼72% of ovules contain coils of pollen tubeswithin the female gametophyte [15]. Synergid cells targetedby these mutant pollen tubes fail to degenerate normally,suggesting a loss of communication between pollen tube andsynergid cells during pollen tube reception [15]. Furthermore,myb triple mutant pollen tubes fail to burst and release sperm.These data suggest that MYB97, MYB101 and MYB120 arecritical for the pollen tube to exchange signals with the femalegametophyte required for successful fertilization.

Pollen tube reception signalling by thefemale gametophytePollen tube reception requires a number of synergid-expressed genes including FER (FERONIA) [16], NTA(NORTIA) [17] and LRE (LORELEI) [18] (Figure 2A).Loss-of-function mutations in of each of these genescause the same phenotype: wild-type pollen tubes coilin mutant ovules and fail to release sperm. FER isa transmembrane receptor-like kinase of the CrRLK1Lfamily predicted to have pollen tube or female-gametophyteligand(s) important for pollen tube reception. FER has amalectin-like extracellular domain, so its ligand could bea carbohydrate or glycoprotein [19] (Figure 2A). FER islocalized to the filiform apparatus, and is required forNTA, a seven-pass transmembrane mildew-resistance locus

C©The Authors Journal compilation C©2014 Biochemical Society

342 Biochemical Society Transactions (2014) Volume 42, part 2

Figure 2 Subgroup 18 (S18) MYB-regulated gene classes and their putative roles during pollen tube receptions

(A) Schematic diagram of the pollen tube and female gametophyte at the beginning of pollen tube reception. Pollen tube

contact with one of the synergid cells is indicated (broken line). Proteins with roles in pollen tube reception and MYB-regulated

gene products are highlighted. Abbreviations: sn, synergid nucleus; vn, vegetative nucleus. (B and C) Schematic diagram

of pollen tube reception between a pollen tube and the synergid that will degenerate. Cytosolic Ca2 + concentrations are

symbolized with a colour spectrum (red, high Ca2 + ; green, low Ca2 + ). (B) FER-dependent NTA relocalization occurs after

pollen tube arrival. (C) Synergid degeneration is accompanied by loss of synergid nuclear integrity. (D) Pollen tube burst,

sperm release and double fertilization.

O family protein, to be relocalized from the secretorysystem to the filiform apparatus upon pollen tube arrival [17](Figures 2A and 2B). NTA may interact with synergid- orpollen-tube-expressed proteins upon relocalization and alsocontains a calmodulin-binding domain in its cytoplasmic C-terminus, possibly allowing synergid cell perception of Ca2 +

oscillations during reception [20] (Figures 2B–2D). LREencodes a glycosylphosphatidylinositol-anchored proteinpredicted to be associated with the synergid membrane [18](Figure 2A). The mechanisms by which FER, NTA and LREdirect pollen tube reception are unknown, and it will beimportant to define how they sense pollen tube arrival toinitiate synergid degeneration and pollen tube burst.

Relatively little is known about the pollen-tube-expressedgenes involved in pollen tube reception, and no directconnections between pollen tube and synergid genes havebeen made. Interestingly, a pair of pollen-tube-expressedmembers of the FER family members [CrRLK receptor-like kinases, ANX1 (ANXUR1) and ANX2 (ANXUR2)]may be negative regulators of pollen tube burst. anx1,anx2double mutants produce pollen tubes that burst very soonafter germination [21,22]. Like FER, the ligands for ANX1and ANX2 are not yet known, and the nature of ANXcontribution to pollen tube reception is unclear because the

double mutant phenotype is not informative about their roleduring synergid interactions.

ACA9 (autoinhibited Ca2 + -ATPase 9) encodes a pollen-specific calmodulin-binding Ca2 + pump localized to thepollen tube plasma membrane [23]. aca9 pollen tubes havegrowth defects; however, they can reach ovules in the upperportion of the pistil and ∼50% of these enter the ovulemicropyle and arrest, but fail to burst [23]. This resultsuggests that ACA9 is important for regulating pollen tubeCa2 + dynamics required for pollen tube burst. Recent live-imaging experiments have shown that Ca2 + concentrationsspike in the pollen tube and the synergids as pollen tubeburst occurs [20] (Figures 2B–2D). It will be very interestingto determine how aca9 pollen and nta (which containsa predicted calmodulin-binding domain) synergid mutantsaffect these dynamics.

Do MYB target genes interact with thepollen tube reception signallingcomponents?myb97,myb101,myb120 triple mutant pollen tubes fail tostop growing and burst within the female gametophyte,so the genes regulated by these transcription factors are

C©The Authors Journal compilation C©2014 Biochemical Society

Regulation of Fertilization and Early Seed Development 343

Figure 3 Pollen tube reception is defective in interaccession and interspecies crosses

(A–H) A. thaliana male sterile 1 (ms1) pistils from the Landsberg accession were pollinated with various pollen donors for

24 h before imaging pollen tube growth patterns using Aniline Blue staining and fluorescence microscopy (as in [32]). m,

micropyle; arrowheads indicate pollen tubes. (A) A. thaliana Col-0 accession, normal entry. (B) A. thaliana myb120-3, coiling.

(C) A. thaliana myb97-1,120-3, coiling and attraction of two pollen tubes. (D) A. thaliana myb97-1,101-4,120-3, coiling. (E)

A. thaliana Cvi-0 accession, two pollen tubes. (F) A. thaliana Lc-0 accession, coiling, two pollen tubes. (G) A. korshinskyi,

coiling, two pollen tubes. (H) O. pumila, coiling, two pollen tubes. (I–L) Emasculated O. pumila pistils were crossed with A.

thaliana Col-0 (I and J) or myb triple mutant (K and L). A. thaliana Col-0 tubes show (I) wild-type pollen tube entry, and (J)

wild-type entry and one tube failing to enter the micropyle (m). myb triple mutants show (K) pollen tube coiling and (L)

failure to enter the micropyle (three tubes) in interspecific crosses. (M) Quantification of pollen tube reception. Arabidopsis

accessions: Col-0 (CS1092), Ler-0 (CS20), Lc-0 (CS28443), Cvi-0 (CS902), Pn-0 (CS28645), Ove-0 (CS28590). Related species:

A. korshinskyi (Ak) (CS4653) or O. pumila (Op) (CS3700). *P < 0.05 (Student’s t-test).

candidates for pollen tube components of the pollen tubereception mechanism. By comparing the transcriptomes ofpistils pollinated with either wild-type or myb triple mutantpollen, three main categories of MYB-regulated genes wereidentified: transporters, small proteins and peptides, andcarbohydrate-active enzymes [15] (Figure 2A). Many of thesegenes were found to be in large gene families, potentiallyexplaining why extensive genetic screening in A. thaliana hasnot identified male pollen tube reception mutants.

Several sugar/proton symporters in the major facilitatorfamily [24] were identified as being MYB-regulated. AtSUC7(At indicates A. thaliana), AtSUC8 and AtSUC9 are highly

induced in wild-type pollen tubes grown in SIV conditions,but are absent from myb triple mutants [15]. myb triplemutants have no pollen tube growth defect in vitro or inthe pistil, suggesting that these sucrose transporters are notrequired to import sugars to fuel growth, but may have aspecialized function in regulating the pollen tube osmoticstate during pollen tube reception.

Multiple small proteins and peptides were found tobe potential targets of MYB activation during pollentube growth (Figure 2A). One of these is a thionin, an87-amino-acid secreted cysteine-rich protein (CRP2460)[25]. Thionins have been shown to nucleate membrane

C©The Authors Journal compilation C©2014 Biochemical Society

344 Biochemical Society Transactions (2014) Volume 42, part 2

pores in artificial lipid bilyers and rat neuronal cells [26];this is an intriguing potential function for pollen-tube-expressed thionins given that myb triple mutant pollentubes fail to express these peptides and also have defects inpollen tube burst and initiating synergid degeneration [15].Interestingly, small (130–152 amino acids) secreted proteinswith domains homologous with stigmatic Papaver rhoeasSI (self-incompatibility) protein PrsS1 [27] were also foundto require pollen tube MYBs for expression in the pollentube [15]. In Papaver, PrsS1 is secreted from the stigmaand is bound by PrpS, its pollen-expressed receptor [28].Ligand–receptor interaction blocks germination of self pollenthrough a mechanism involving Ca2 + as a second messengerand PCD (programmed cell death) [29]. The function of A.thaliana SPHs (S-protein homologues) is unclear [30] and itis surprising to find that they are expressed in pollen tubes inresponse to growth through the pistil. It will be interestingto test whether a signalling module similar to that describedin Papaver is involved in gametophyte interactions that leadto synergid degeneration and pollen tube burst.

The third category of pollen tube MYB-regulated genesencode proteins that interact with carbohydrates, many ofwhich are predicted to be extracellular (Figure 2A). Theseinclude hydrolases (i.e. glycoamylase, O-glycosylhydrolase,β-1,3-glucanase, pectin lyase-like) and pectin methylesteraseinhibitors [15]. These proteins may be expressed by the pollentube to modify the cell wall in preparation for pollen tubereception. Alternatively, these proteins may modify the cellwall or other pollen-derived carbohydrates for recognition bythe female gametophyte. As mentioned above, FER containsan extracellular malectin domain that could interact witha pollen-tube-derived carbohydrate, and, since myb triplemutant pollen tubes behave as though FER signalling isdefective, it will be interesting to determine whether pollentube MYBs regulate production of FER ligands.

MYB-mediated pollen tube differentiationis important for species recognition duringpollen tube receptionPollen tube reception fails in interspecific crossesof Rhododendron [31] or A. thaliana [16]; pollen tubes ofone species overgrow without releasing sperm in ovulesof the exotic species. These data suggest that pollen tubereception is an important pre-zygotic barrier to interspecifichybridization. When A. thaliana myb triple mutant pollentubes enter a wild-type A. thaliana ovule, they overgrowand fail to release sperm [15]. These observations led to thehypothesis that pollen tube MYBs may control expression ofthe determinants of pollen tube identity during interspecificrecognition.

To begin to assess the role of pollen tube MYBs indetermining pollen tube recognition, pollen tube receptionwas investigated in interaccession and interspecies crosses(Figure 3). The rates of pollen tube overgrowth when mybtriple and myb double mutants were used to pollinate A.

thaliana pistils were higher than in any of the interaccessioncrosses tested (Figure 3M). Arabidopsis korshinskyi andOlimarabidopsis pumila pollen tubes overgrew in A. thalianaovules more frequently than any of the accessions tested, butthe A. thaliana myb triple mutant phenotype was even morepronounced (Figure 3M). These results are consistent withthe idea that pollen tube MYBs control pollen tube identityas recognized by the female gametophyte.

When A. korshinskyi or O. pumila were used as femalesin crosses with A. thaliana pollen, increased rates of pollentube overgrowth were not observed because the percentageof untargeted ovules (probably due to incongruity in pollentube attraction) was so high that pollen tube reception couldnot be adequately assessed (Figures 3I, 3J and 3M). However,crosses of A. thaliana myb triple mutant pollen on to A.korshinskyi or O. pumila pistils showed increases in ratesof pollen tube coiling as well as increases in the number ofuntargeted ovules (Figures 3K–3M). This finding suggeststhat MYBs are required for two levels of pollen tube identity:(i) between species, and (ii) within species.

Future work will need to address the mechanisms by whichindividual MYB target genes confer these identities. Onepossibility is that MYB-mediate pollen tube differentiationfails when pollen tubes grow through an exotic pistil.Alternatively, MYBs may be properly activated by the exoticpistil and may in turn activate their targets, but the pro-ducts of these target genes are not recognized by the exoticfemale gametophyte. In either case, exploration of how thepistil induces MYB97, MYB101 and MYB120 and howthe products of their target genes mediate gametophyteinteractions provides a path to understand how suitable matesare identified during flowering plant reproduction.

Funding

This work was supported by the National Science Foundation [grant

number IOS-1021917]. A.R.L. was supported by a National Institutes

of Health Training in Molecular and Cellular Biochemistry Grant [grant

number T32 GM007601] (PI: Kimberly L. Mowry).

References1 Johnson, M.A. and Preuss, D. (2002) Plotting a course: multiple signals

guide pollen tubes to their targets. Dev. Cell 2, 273–2812 Dresselhaus, T. and Franklin-Tong, N. (2013) Male–female crosstalk

during pollen germination, tube growth and guidance, and doublefertilization. Mol. Plant 6, 1018–1036

3 Hamamura, Y., Saito, C., Awai, C., Kurihara, D., Miyawaki, A., Nakagawa,T., Kanaoka, M.M., Sasaki, N., Nakano, A., Berger, F. and Higashiyama, T.(2011) Live-cell imaging reveals the dynamics of two sperm cells duringdouble fertilization in Arabidopsis thaliana. Curr. Biol. 21, 497–502

4 Beale, K.M., Leydon, A.R. and Johnson, M.A. (2012) Gamete fusion isrequired to block multiple pollen tubes from entering an Arabidopsisovule. Curr. Biol. 22, 1090–1094

5 Kasahara, R.D., Maruyama, D., Hamamura, Y., Sakakibara, T., Twell, D.and Higashiyama, T. (2012) Fertilization recovery after defective spermcell release in Arabidopsis. Curr. Biol. 22, 1084–1089

C©The Authors Journal compilation C©2014 Biochemical Society

Regulation of Fertilization and Early Seed Development 345

6 Maruyama, D., Hamamura, Y., Takeuchi, H., Susaki, D., Nishimaki, M.,Kurihara, D., Kasahara, R.D. and Higashiyama, T. (2013) Independentcontrol by each female gamete prevents the attraction of multiplepollen tubes. Dev. Cell 25, 317–323

7 Kessler, S.A. and Grossniklaus, U. (2011) She’s the boss: signaling inpollen tube reception. Curr. Opin. Plant Biol. 14, 622–627

8 Palanivelu, R. and Johnson, M.A. (2010) Functional genomics of pollentube–pistil interactions in Arabidopsis. Biochem. Soc. Trans. 38, 593–597

9 Higashiyama, T., Kuroiwa, H., Kawano, S. and Kuroiwa, T. (1998)Guidance in vitro of the pollen tube to the naked embryo sac of Toreniafournieri. Plant Cell 10, 2019–2032

10 Palanivelu, R. and Preuss, D. (2006) Distinct short-range ovule signalsattract or repel Arabidopsis thaliana pollen tubes in vitro. BMC Plant Biol.6, 7

11 Okuda, S., Tsutsui, H., Shiina, K., Sprunck, S., Takeuchi, H., Yui, R.,Kasahara, R.D., Hamamura, Y., Mizukami, A., Susaki, D. et al. (2009)Defensin-like polypeptide LUREs are pollen tube attractants secretedfrom synergid cells. Nature 458, 357–361

12 Okuda, S., Suzuki, T., Kanaoka, M.M., Mori, H., Sasaki, N. andHigashiyama, T. (2013) Acquisition of LURE-binding activity at the pollentube tip of Torenia fournieri. Mol. Plant 6, 1074–1090

13 Liu, J., Zhong, S., Guo, X., Hao, L., Wei, X., Huang, Q., Hou, Y., Shi, J.,Wang, C., Gu, H. and Qu, L.J. (2013) Membrane-bound RLCKs LIP1 andLIP2 are essential male factors controlling male-female attraction inArabidopsis. Curr. Biol. 23, 993–998

14 Qin, Y., Leydon, A.R., Manziello, A., Pandey, R., Mount, D., Denic, S.,Vasic, B., Johnson, M.A. and Palanivelu, R. (2009) Penetration of thestigma and style elicits a novel transcriptome in pollen tubes, pointing togenes critical for growth in a pistil. PLoS Genet. 5, e1000621

15 Leydon, A.R., Beale, K.M., Woroniecka, K., Castner, E., Chen, J., Horgan, C.,Palanivelu, R. and Johnson, M.A. (2013) Three MYB transcription factorscontrol pollen tube differentiation required for sperm release. Curr. Biol.23, 1209–1214

16 Escobar-Restrepo, J.M., Huck, N., Kessler, S., Gagliardini, V., Gheyselinck,J., Yang, W.C. and Grossniklaus, U. (2007) The FERONIA receptor-likekinase mediates male–female interactions during pollen tube reception.Science 317, 656–660

17 Kessler, S.A., Shimosato-Asano, H., Keinath, N.F., Wuest, S.E., Ingram, G.,Panstruga, R. and Grossniklaus, U. (2010) Conserved molecularcomponents for pollen tube reception and fungal invasion. Science 330,968–971

18 Capron, A., Gourgues, M., Neiva, L.S., Faure, J.E., Berger, F., Pagnussat,G., Krishnan, A., Alvarez-Mejia, C., Vielle-Calzada, J.P., Lee, Y.R. et al.(2008) Maternal control of male-gamete delivery in Arabidopsis involvesa putative GPI-anchored protein encoded by the LORELEI gene. Plant Cell20, 3038–3049

19 Lindner, H., Muller, L.M., Boisson-Dernier, A. and Grossniklaus, U. (2012)CrRLK1L receptor-like kinases: not just another brick in the wall. Curr.Opin. Plant Biol. 15, 659–669

20 Iwano, M., Ngo, Q.A., Entani, T., Shiba, H., Nagai, T., Miyawaki, A., Isogai,A., Grossniklaus, U. and Takayama, S. (2012) Cytoplasmic Ca2 + changesdynamically during the interaction of the pollen tube with synergid cells.Development 139, 4202–4209

21 Miyazaki, S., Murata, T., Sakurai-Ozato, N., Kubo, M., Demura, T., Fukuda,H. and Hasebe, M. (2009) ANXUR1 and 2, sister genes to FERONIA/SIRENE, are male factors for coordinated fertilization. Curr. Biol. 19,1327–1331

22 Boisson-Dernier, A., Roy, S., Kritsas, K., Grobei, M.A., Jaciubek, M.,Schroeder, J.I. and Grossniklaus, U. (2009) Disruption of thepollen-expressed FERONIA homologs ANXUR1 and ANXUR2 triggerspollen tube discharge. Development 136, 3279–3288

23 Schiott, M., Romanowsky, S.M., Baekgaard, L., Jakobsen, M.K., Palmgren,M.G. and Harper, J.F. (2004) A plant plasma membrane Ca2 + pump isrequired for normal pollen tube growth and fertilization. Proc. Natl. Acad.Sci. U.S.A. 101, 9502–9507

24 Reinders, A., Sivitz, A.B. and Ward, J.M. (2012) Evolution of plant sucroseuptake transporters. Front. Plant Sci. 3, 22

25 Silverstein, K.A., Moskal, Jr, W.A., Wu, H.C, Underwood, B.A., Graham,M.A., Town, C.D. and VandenBosch, K.A. (2007) Small cysteine-richpeptides resembling antimicrobial peptides have been under-predictedin plants. Plant J. 51, 262–280

26 Hughes, P., Dennis, E., Whitecross, M., Llewellyn, D. and Gage, P. (2000)The cytotoxic plant protein, β-purothionin, forms ion channels in lipidmembranes. J. Biol. Chem. 275, 823–827

27 Foote, H.C., Ride, J.P., Franklin-Tong, V.E., Walker, E.A., Lawrence, M.J.and Franklin, F.C. (1994) Cloning and expression of a distinctive class ofself-incompatibility (S) gene from Papaver rhoeas L. Proc. Natl. Acad.Sci. U.S.A. 91, 2265–2269

28 Wheeler, M.J., de Graaf, B.H., Hadjiosif, N., Perry, R.M., Poulter, N.S.,Osman, K., Vatovec, S., Harper, A., Franklin, F.C. and Franklin-Tong, V.E.(2009) Identification of the pollen self-incompatibility determinant inPapaver rhoeas. Nature 459, 992–995

29 Poulter, N.S., Wheeler, M.J., Bosch, M. and Franklin-Tong, V.E. (2010)Self-incompatibility in Papaver: identification of the pollenS-determinant PrpS. Biochem. Soc. Trans. 38, 588–592

30 Ride, J.P., Davies, E.M., Franklin, F.C. and Marshall, D.F. (1999) Analysis ofArabidopsis genome sequence reveals a large new gene family inplants. Plant Mol. Biol. 39, 927–932

31 Williams, E.G., Kaul, V., Rouse, J.L. and Palser, B.F. (1986) Overgrowth ofpollen tubes in embryo sacs of Rhododendron following interspecificpollinations. Aust. J. Bot. 34, 413–423

32 Mori, T., Kuroiwa, H., Higashiyama, T. and Kuroiwa, T. (2006) GENERATIVECELL SPECIFIC 1 is essential for angiosperm fertilization. Nat. Cell Biol. 8,64–71

Received 24 September 2013doi:10.1042/BST20130223

C©The Authors Journal compilation C©2014 Biochemical Society