The study of how pollen tubes are successfully guided to target ovules through a combination of mechanistic techniques alongside the reception of chemical attractants is intensely researched.
Imaging advances of pollen tube growth through new techniques, model organisms and new methods of high-throughput genetic analysis have allowed this process to become comprehensively understood. This review discusses the recent research done in the field and the genes involved. Issues remain with dynamic imaging of the pollen tube guidance process and lack of systemic approaches to understanding interactions between the individually characterised peptides and genes implicated. Introduction:Figure 1: Adapted from(1) left image shows depiction of the germinated pollen tube, the 4 pictures on right depict electron micrograph imaging of pollen grains in various stages of germination. The success of the sexual reproduction process relies on effective crosstalk between the female and male gametes. In angiosperms, gametes are contained within embryo sacs and inside pollen grains. (2)Male gametes must be guided to the female ovule via germination and guided growth of pollen tubes. (3,4)Pollen tube guidance requires the ability to accurately receive and interpret signals secreted by female gametes.
(5) This procedure is divided into competency control for each pollen tube, the successful termination of the pollen tube and termination of pollen tube guidance mechanisms once the tube has reached its destination. Pollen tube guidance can be broken down into two stages: The preovular guide stage and the ovular guide stage.(6) These two stages will be reviewed. Preovular GuidanceGeneSex ExpressionProtein FamilyFunctionNo Transmitting Tract (NTT)Female Transcription FactorTransmitting tract developmentSCAFemale LTP-Like CRPPollen Tube AdhesionCLE45FemaleCLVE/ESR RelatedPollen Tube GrowthSKM1/2FemaleLRR-RLKReceptor for CLE45LUREFemale CRPMicropylar GuidanceTable 1: Identified genes involved in the Pollen tube guidance process written about in this review. Above Genes implicated in Preovular guidance.
Pollen tubes must first penetrate the intercellular area within the stigma and style, they then have to proceed along a nutrient-rich extracellular matrix of transmitting tract tissues (TT) moving to the ovary. TT are diverse between different species.(7,8,9)Different female components, such as stimulants for the germination of pollen and pollen tube growth, are supplied to supplement growth towards the ovary.(6) The transmitting tract is the common denominator between different plant varieties when considering mechanistic differences between species. It’s indispensable in Arabidopsis Thaliana.
An identified gene in Transmitting tract development, the NTT (No Transmitting Tract) gene, when knocked out – resulted in pollen tubes moving slower through the TT or terminating prematurely. (8) There is a clear distinction that while pistils, are similar between species, their morphological differences and tissue specifics between the surface of the stigma and the ovule contribute largely to a self/non-self-identification system. It supports directional control, rapid growth and competency regulation of the pollen tube.(5)(10)(8,11,12)CLE45 is a peptide localised in the pistil of Arabidopsis. The synthetically produced CLE45 peptide is a promoter of pollen tube growth in vitro. It interacts with repeat receptor-like kinases; SKM1 and SKM2 (13). A double mutant affecting O-arabinosyltransferase, resulted in pollen tube growth arrest.
(14) This suggests that there are other O-arabinosylated peptides in correct pollen tube growth systems.PSK (Phytosulfokine) is a disulfate pentapeptide with proliferation properties. (15) If added to a culture media, pollen tube growth will accelerate in vitro. (16) If Tyrosylprotein sulfotransferase (TPST), which causes the sulfonylation of PSK (or the PSK receptor- PSKR) is knocked down, there is a reduction in fertility due to pollen tube aberrations in the pistil. (15).Pollen tubes emerge in Torenia species from the opposing terminus of the dissected style after germinating at the proximal end of the style. When germinating in a section at the centre of the style tissue, pollen tubes develop in both directions at a comparable frequency. It’s possible that a chemotropic directional regulator isn’t present in the styles of Torenia plants, and pollen tubes are able to grow directionally along the TT towards the ovule once entry into the style has occurred.
(17)Ovules sampled from the ovary are grown and cultivated in pollen growth media to allow pollen tubes to develop within the cut and pollinated style. The pollen tubes are attracted by the ovule and double fertilisation occurs in the ovule. When the pollen granules are simply added to cultivated media and germinated, little to no ovule attraction occurs. There is good empirical evidence here to suggest that female sporophytic tissues are likely to increase the competency and attraction of pollen tubes to be able to respond to an attractant mechanism. (18,19)This means pollen tubes require an activating step for development to occur within the pistil section exact pollen tube guidance to occur. A connection was identified between the growth stage of the pollen tube and its attraction for chemical ‘ovular-attractants’ in the plant species T. fournieri. T.
Fournieri is unusual amongst plants as it has an exposed embryo sac which make it an ideal model species for researching the role of pollen guidance systems(18)(20). LURE peptides are designated as pollen tube ‘ovular-attractants’ (LURE 1, LURE2) and are members of a defensin subclass of peptides rich in cysteine. Using immune-staining techniques it was shown that the in-vitro pollen tubes germinated on the PGM (pollen growth media) had no response to a LURE2 peptide even 12 hours after germination. LURE peptides consistently failed to bind to the pollen tubes. Developing through a style with a certain length beyond a predetermined threshold could be key to giving competency to developing pollen tubes in the model T.Fournieri. Tubes grown through more diminutive styles were able to bind LURE2 attractants but did not behave as if they were attracted. We conclude there is a difference between the act of binding and subsequent growth response – they may be separate independent stages.
The physiological alterations detected are most likely evidence of pollen tube developmental maturation.(20) Ovular GuidanceGeneSex ExpressionProtein FamilyFunctionCHX21/23Male Cation/proton exchangersMicropylar GuidanceMYB98FemaleMYBFiliform Apparatus DevelopmentTcLURE/AtLureFemale CRPsPollen Tube AttractantPRK6MaleRLKReceptor Kinase for Female Ovule SignalTable 2: Genes implicated in Ovular GuidanceThe move of pollen tubes from the TT and entry to the micropyle in a well-studied process. (12)(10) and has led to the identification of several peptide pollen tube attractants. Functional Female gametophytes play an important role in attracting the pollen grains to the ovule. This leads us to the assumption that attractants must be made up of constituents of female gametophytes. However, some components of long-distance attraction that have not yet been characterised (21)(22)Identified guidance signals include cation and proton exchangers (CHX) in Arabidopsis.
CHX21 and CHX23 were found to be critical for pollen tube guidance. Single mutations in CHX21 and CHX23 had no effect, but fertility was damaged in double mutants. Double mutant pollen successfully germinated and developed through the TT but failed to slow down and re-orientate to reach the ovule. These mutants also did not succeed in entering the micropyle of the sampled ovules.(23) The system of study of Torenia and the technique of semi in-vitro study revealed synergid cells, which are two uniquely functional cell units located nearby to the ovary(24). Exiting from the TT, the pollen tube is directed to the micropyle, the tube moves into a single synergid cell which starts to decay as the tube enters it. The tube stops growing, ruptures to release its contained sperm cells, which subsequently move to the egg and central cell and fertilise them. These cells were proven to be necessary for pollen tube guidance(25) Figure 2 adapted from (26) depiction of the crucial components of the female gametophyte There is transcriptional regulatory function in the R2R3 Myb protein family.
(24) MYB98 was identified in a screen of Arabidopsis, which regulates the genes localised within synergid cells and are essential for the nascent formation of the filiform apparatus. MYB98 is exclusively localised within synergid cells. MYB98 mutant’s phenotypes display aberrations with distinct processes; pollen guidance and improper formation of the filiform apparatus. It was observed that the MYB98 promoter was localised totally in the nucleus of the synergid cell.
(25)Figure 3 adapted from (26): demonstrating localisation of GFP fusion MYB98 in the synergid cell nucleus (SCN). In the panel on the right, localisation in the filiform apparatus is visible. (FA) SC = Synergid cell.LURE1 and LURE2 polypeptides attracted pollen tubes in vitro in a concentration dependent manner. The LUREs have two distinct characteristics; their sequence diversity and their preference for species. The TcLure1 (TcCRP1) which is orthologous to TfCRP1, from the CRP family, from the species T.
concolor (a species related to T. fournieri), was a pollen attractant utilised as a concentration gradient, but didn’t attract as many pollen tubes from other species. A hypothesis has been formed that while the attractants TfCRP1 and TcCRP1 are orthologous, high sequence diversity allows plant ovules to maintain species-specific pollen tube guidance.
(28) The specificity of each of these Torenia species LURE proteins is further demonstrated in a 2006 paper utilising T. fournieri and 4 other species (Torenia baillonii, Torenia concolor, Lindernia Vandellia crustacea, and Lindernia micrantha). Closely related species but encompassing two genera, chosen for their exserting embryo sac. Evidence was found that pollen tubes were attracted to synergid cells of the embryo sacs of their respective species at a far greater rate than its non-species equivalent. Providing evidence that there is a species-specific chemical produced in each related synergid cell to allow its species’ pollen tube to recognise it.
(29)A greater problem has been the identification of receptors for all of these signals released by the ovule. The family of receptors that play a critical role in the male gamete may include leucine-rich RLKs such as the ERECTA family of receptor kinases, it is possible these receptors may be the more direct binding target of LURE proteins. (30) (31) A receptor located in the tip of the pollen tube called receptor-like kinase 6 (PRK6) was a crucial receptor for detecting the LURE1 chemoattractant in Arabidopsis in partial in-vivo conditions. (32) PRK6 has interactions with Rho of plant guanine nucleotide-exchange factors (ROPGEFs(33)(34).
The PRK6 accumulates asymmetrically in favour of an extracellular AtLURE1 source before redirecting pollen tube tip growth. PRK6 is a key receptor for LURE attractants and is involved in the recruitment of pollen tube tip growth machinery.Figure 4 adapted from (32) demonstrating semi-in-vivo pollen tube growth coupled with AtLURE1 response assay in prk knockout mutations approx. 9 hours after pollination. It’s very clear that knocking out prk6 has a dramatic effect on the guidance of pollen tube in the 4th panel although PRK1 and PRK3 also show disrupted response to AtLURE1. This is scaled at 100µm (scale bar).
Termination of SignallingGeneSex ExpressionProtein FamilyFunctionSRNFemale N/APollen Tube RuptureFERFemale RLKPollen Tube RuptureLorelaiFemale GPI-APTransports FERMYBFemale MYBPollen Tube OvergrowthEC1Male CRPPlasmogamyGEX2Male Membrane ProteinPlasmogamyFIS2Female Polycomb ComplexPlasmogamyTable 3: Genes implicated in Termination of signallingRegulation of pollen tube reception is poorly comprehended. (35) Research showed a partially sterile mutant of the female gametophyte was isolated from Arabidopsis, called Feronia, which was found to affect the process. In this mutant, sac and guidance development were not affected in all of the ovules. However, 50% of the ovules contained mutant female gametophytes. When the pollen tube moved into the synergid of the Feronia female gametophyte – It did not stop growing, failing to halt and rupture, and releasing its sperm cells.
It was thus concluded that the Feronia mutation disrupts this termination signal from being either released or received by the male pollen tube. Upon analysis of mutant synergid formation with GUS markers, it was revealed that designation and differentiation of the synergids were as usual. The expression of GUS remained consistently high in synergid cells long after pollen tube entry, whereas in wild-type, it decreased rapidly at this point. This leads us to believe that the Feronia mutation leaves the expression of synergid specific genes switched ‘on’ for far longer than it should normally be for normal pollen tube guidance to occur.
(35) Feronia has since been found to intersect with several major plant signalling processes. FER is broadly expressed in plants with the exception of pollen.(36) Involved with auxin pathway (36), abscisic acid (37), brassinosteroid, and ethylene. (38)(39) Interactions have been shown to occur with rapid alkinisation factor 1 (RALF1)(40). Mutants of FER are thus pleiotropic with many consequences caused as a result of affecting its ability to function. FER additionally regulates susceptibility to powdery mildew fungus (41) and is involved in mechanical signal transduction (42).
It may be possible that FER modulates unique signalling under varying cellular and developmental factors and environmental changes.Further research revealed FER protein appears to gather asymmetrically within the membrane of the synergid cells at the filiform apparatus, plays a strong role in female-controlled pollen tube reception and may even feature in reproductive isolation barriers(43)FER codes a receptor kinase localised on the synergid cells. The FER signalling pathway is necessary for starting and adjusting calcium responses and for coupling the programmed cell death of one of the synergid cells.
The calcium concentrations have been found to be interchangeable between both synergids, which leads us to believe that their ultimate destiny of death and survival are decided by interactions that are reversible with Pollen Tubes. (44)Lorelei is another mutation reminiscent of the Feronia mutant, a mutant in Arabidopsis impairing the release of sperm cells. Pollen tubes that reach the lorelei embryo sacs often fail to rupture and continue growing aberrantly within the embryo sac. Additionally, lorelei embryo sacs consistently attract more tubes post arrival of the first primary pollen tube. The lorelei gene is also localised in the synergid cells before fertilization and encodes a small specific putative glycosylphosphatidylinositol-anchored protein (GAP).
The observations lend credence to the concept that signalling pathways at the synergid membrane play an important role in regulating male sperm cell release. Although there is evidence to suggest that GAPs play vital roles in initiation of fertilization in mammals,(45)(46) angiosperms have seemingly evolved a reproductive system that utilises the unique features of these same proteins in a not dissimilar context.(47) Polyspermy in lre MutantsTable 4: adapted from (47) demonstrating the increased amount of polyspermy occurring in aberrant mutants with Lorelei mutationsPolytubey is a useful mechanism that helps to limit the delivery of excessive numbers of sperm cells (beyond a single pair) to an ovule, it also supplies a method of ‘saving’ fertilization in ovules that have been the recipients of dysfunctional sperm. It helps to provide a greater chance of success in reproduction by sending pollen tubes to every ovule.(48)(49).If initial fertilisation fails, the remaining synergid cell can continue signalling for further pollen tubes in the hopes of successfully completing the fertilisation process. If a failure occurs a second time, there will be no further signalling as there are no viable synergid cells left.
(49)Termination of SignalCombinations of membranes of both the endosperm and active synergid cell, demonstrate a unique form of cell fusion within the well-categorised egg cell – sperm cell and central cell-sperm fusion. This observation has given credence to the theory that cell fusion functions to de-activate cells and is involved in rapid signal termination (50)(Figure 5 adapted from D. Maruyama, 2015) depicting ‘cell fusion’ that results in termination of pollen tube signalling.ConclusionsOur understanding of the pollen tube guidance process has grown massively in the last 10 years with the advent of greater observational and experimental techniques such as pollination-enriched translatomic analysis (51).
It’s the opinion of this author that while many proteins and genes are involved in the process of signalling, the overall mechanistic pathways and intracellular interactions have yet to be described adequately. This approach would require a more systemic biological approach which is likely to be difficult due to its ambitious scope. Not a lot is known about how the networks and cell expansion machinery are integrated together (52)Another aspect to the question is that the component that allows pollen tube cells the competency to respond to signals from LUREs is unknown and upstream molecules for guidance that could operate prior to LUREs in the pathway are also unknown.It is possible that important individual components are still unknown, including factors like repellents and long-distance attractants. Pollen tube guidance is a very dynamic process which necessitates live imaging. ‘ex vitro’ on a model plant due to its external embryo sac which has allowed us to make many more discoveries. New tech such as two-photon excitation confocal microscopy could give us the toolset to reveal interactions inside live pistil tissue(53)(54).
This approach combined with a systemic research method should allow us to further understand the pollen tube guidance event.