Male gametophyte development and function in angiosperms

21 Pages·2016·2.15 MB·English

by  Said Hafidh

PlantReprod
DOI10.1007/s00497-015-0272-4
REVIEW
Male gametophyte development and function in angiosperms:
a general concept
Said Hafidh1 • Jan F´ıla1 • David Honys1,2
Received:12September2015/Accepted:19December2015
(cid:2)Springer-VerlagBerlinHeidelberg2016
Key message Overview of pollen development. describe the recent advances in regulatory mechanisms
Abstract Malegametophytedevelopmentofangiosperms involved such as posttranscriptional regulation (including
is a complex process that requires coordinated activity of masstranscriptstorage)andposttranslationalmodifications
different cell types and tissues of both gametophytic and to modulate protein function, intracellular metabolic sig-
sporophytic origin and the appropriate specific gene naling,ionicgradientssuchasCa2?andH?ions,cellwall
expression. Pollen ontogeny is also an excellent model for synthesis, protein secretion and intercellular signaling
thedissectionofcellularnetworksthatcontrolcellgrowth, within the reproductive tissues.
polarity, cellular differentiation and cell signaling. This
article describes two sequential phases of angiosperm Keywords Pollen development (cid:2) Male gametophyte (cid:2)
pollen ontogenesis—developmental phase leading to the Pollen tube growth (cid:2) Flowering plants
formation of mature pollen grains, and a functional or
progamicphase,beginningwiththeimpactofthegrainson
the stigma surface and ending at double fertilization. Here Introduction
we present an overview of important cellular processes in
pollen development and explosive pollen tube growth Reproductionisacharacteristicfeatureoflivingorganisms
stressing the importance of reserves accumulation and ensuring the continuity of life on earth through a series of
mobilization and also the mutual activation of pollen tube successive generations. In plants, there is an alternation of
and pistil tissues, pollen tube guidance and the communi- sexual and asexual generations, sporophyte and gameto-
cationbetweenmaleandfemalegametophytes.Wefurther phyte. In the sporophyte, the individuals consist of diploid
cells and reproduce asexually through haploid spores gen-
erated by the meiotic division in sporogenous tissue. The
CommunicatedbyEnricoSchleiff. spores germinate into the gametophyte, where specialized
reproductiveorgans,femalearchegoniaandmaleantheridia
Acontributiontothespecialissue‘Pollendevelopmentandstress
areformedinwhichhaploidgametesofseparatesexesarise.
response’.
Thefusionofmaleandfemalegametesleadstotheforma-
Electronicsupplementarymaterial Theonlineversionofthis tionofadiploidzygote,thefirstcellofthenextsporophyte
article(doi:10.1007/s00497-015-0272-4)containssupplementary
generation. In the evolutionary line of vascular plants
material,whichisavailabletoauthorizedusers.
(Tracheophyta), gametophytic reduction and increased
& DavidHonys functional dependence on the sporophyte is apparent.
[email protected] Angiosperms, which currently account for more than
280,000species(reviewedbyScotlandandWorltey2003),
1 InstituteofExperimentalBotanyASCR,v.v.i.,Rozvojova´
constitutetheoverwhelmingmajorityofplantspecieswith
263,16500Prague6,CzechRepublic
maximum reduction of the gametophyte. This reduction
2 DepartmentofExperimentalPlantBiology,Facultyof
togetherwithprotectionofthereproductiveorganswithina
Science,CharlesUniversityinPrague,Vinicˇna´ 5,
12844Prague2,CzechRepublic flower and stringent selection of the fittest pollen to
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reproduce is considered to be the main cause ofthe evolu- thepollentubesexplosivegrowththatmakestheprogamic
tionary success of angiosperms (Mulcahy 1979; Mulcahy phase interesting but also the mutual activation of pollen
et al. 1996). Most angiosperms form bisexual flowers con- tube and pistil tissues, pollen tube guidance and the com-
taining anthers and pistils, where both male and female munication between the two gametophytes (reviewed by
gametophytes develop. Both structures—pollen grain and Dresselhaus and Franklin-Tong 2013).
ovule—aremicroscopic,consistingofonlyafewcellsfully Unlikethesporophyte,themalegametophyterepresents
supportedbythesurroundingsporophytictissuesandorgans highly reduced, two- or three-celled model system provid-
fortheirdevelopment.Theonlytime,whengametophytesof ing a unique opportunity to study the developmental regu-
flowering plants exist independently of the sporophyte, is lation of cell morphogenesis and differentiation at many
whenmaturepollenisshedfromtheantherandiscarriedto levels as well as the functional interactions between dif-
thestigmatoundergoreproductionwithfemalegametes. ferentcelltypes(BergerandTwell2011;Borgetal.2009;
Inthepast,thesmallsizeofthemalegametophytemade Dresselhaus and Franklin-Tong 2013; Dresselhaus and
itsexploration—andevenitsdiscovery—verycomplicated. Sprunck2012;Hafidhetal.2014;KesslerandGrossniklaus
Therefore,theexternalappearanceofpollengrainsstoodat 2011;Ma 2005;Rutleyand Twell 2015; Twell 2011).
the beginning of pollen research (Malpighi 1675, 1679;
Purkyneˇ 1830).Furtherknowledgeofthepollenandpollen
tubestructureandfunctionwasgraduallyacquiredtogether Pollen maturation: get ready for the race!
with the otherbasicphenomena associated with the sexual
reproduction of angiosperms, such as the existence of bi- Diploid microspore mother cells (microsporocytes) are
and tri-cellular pollen (Elfving 1879), differentiation of encapsulatedintheyoungantherloculisurroundedbyfour
vegetative and generative cells (Strasburger 1884) and the cell layers—tapetum, middle layer, endothecium and epi-
double fertilization (Nawaschin 1898). Subsequent con- dermis. The microsporocytes secrete cell wall materials
siderableprogressinpollenresearchhasbeendrivenbythe consisting of b-1,3-glucan and callose. After two meiotic
rapid development of cytological and molecular methods. divisions, the microsporocytes divide into four haploid
Equally important was the introduction of in vitro, semi microspores forming a tetrad (Fig. 1). In Avena sativa,
invivoandinvivotechniquesandinparticulartheonsetof microspore mother cells communicate via cytoplasmic
‘‘high-throughput’’ technologies in the recent 20 years. bridges enabling the synchronization of meiotic divisions
throughout the loculus (Brett and Waldron 1990). The
second meiotic division is followed by the synthesis of
Male gametophyte callose walls between the individual microspores within a
tetrad. However, the timing of callose wall formation dif-
Male gametophyte development of angiosperms is a com- fers between different plant species (Chen and Kim 2009;
plex process that requires coordinated activity of different De Storme and Geelen 2013; Lu et al. 2014).
celltypesandtissuesofbothgametophyticandsporophytic During meiosis, the secretory tapetal cells differentiate
originunderlaidbyspecificgeneexpressionpatterns.Male into binuclear polar cells lacking the primary cell wall,
gametophytedevelopmentiscomprisedoftwoconsecutive especially at the loculi-facing side. These cells contain
phases, developmental and functional. abundantribosomes,mitochondria,endoplasmicreticulum,
The developmental phase takes place in anther loculi Golgi apparatus and specialized lipid-rich organelles,
and leads to the release of mature pollen grains from the tapetosomes(Bedinger1992;HsiehandHuang2005;Ting
anthers. It is characterized by the functional specialization et al. 1998), close to the plasma membrane facing the
of two cell types—vegetative cells and male gametes, the anther loculi. The tapetal cells are interconnected by
sperm cells, which together represent the male germline cytoplasmic bridges allowing the coordination of their
(reviewed by Berger and Twell 2011). This process is activities. Transcriptomic studies of anther tissues in vari-
underpined by two successive cell divisions accompanied ous plant species demonstrated the precise control of the
by remarkable morphological and physiological differen- activity and subsequent programmed cell death of tapetal
tiation of both cell types including the synthesis of spe- cells and the tight coordination of these processes with
cialised pollen cell wall and the storage of protective pollen development (Huang et al. 2011).
substances and metabolic reserves. Young microspores in tetrads undergo rapid develop-
The functional or progamic phase is initiated after the ment accompanied by synthesis of the cell wall consisting
pollen grain lands on the stigma. Pollen is activated by of an inner intine and outer exine. After the emergence of
rehydration, germinates and produces a long pollen tube partially formed exine, microspores are released from tet-
that grows into the pistil tissues. Its growth ends with a rads in a synchronised manner (Fig. 1). The release of the
double fertilization after reaching the ovule. It is not only microspores is prompted by the activity of an enzyme
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Fig.1 Schematicdiagramdescribingpollendevelopment(adaptedandsimplifiedfromHonysetal.2006)
mixture secreted by tapetal cells, with callase (b-1,3-glu- biopolymers and its role is the protection of the pollen
canase) being its essential component responsible for the internal environment (including genetic information) after
callose degradation (Lu et al. 2014; Scott et al. 2004). pollensheddingfromtheanthers.Althoughthebiochemical
Callase synthesis was shown to be controlled by MYB- pathway of sporopollenin synthesis remains elusive, its
family transcription factor AtMYB80 that links pollen synthesis requires close cooperation between microspores
maturation with tapetum development resulting in pro- and tapetum (Ariizumi and Toriyama 2011; Dobritsa et al.
grammed cell death of tapetal cells (Phan et al. 2011; 2009,2010;Quilichinietal.2015).Therecalcitrantexineis
Zhangetal.2007).Propertimingofcallasesecretionisone not distributed evenly around pollen grains; it is not
of the critical moments in microsporogenesis and its dis- deposited or it is reduced in the apertures, where pollen
tortion causes male sterility (Worrall et al. 1992). Free tubesemerge(FurnessandRudall2004).Thenumber,size
microspores rapidly enlarge and numerous small vacuoles anddistributionofaperturesareanimportantclassification
eventually merge into one large vacuole pushing the factor that is under strict sporophytic control. In Ara-
microspore nucleus from its central position to the cell bidopsis,aperturemarkingdependsonthepriorlocalisation
periphery. The actual microspore polarisation is not just a of INAPERTURATE POLLEN1 protein (Dobritsa and
passive event caused by the growth of vacuoles but a Coerper2012; Dobritsa et al. 2011).
highly dynamic process requiring the active participation Formation of the pollen coat is completed in the later
of microtubules (Oh et al. 2010; Park et al. 1998; Twell stages of microgametogenesis when the residues of the
2011). During microsporogenesis, the tapetal cells remain degeneratingtapetumaredepositedonthesurfaceofpollen
highly metabolically active. They secrete proteins, lipids, grains (Quilichini et al. 2015). Pollen coat determines the
carbohydratesandsecondarymetabolitestotheloculitobe pollen adhesiveness, colour, taste and aroma. These prop-
used by developing microspores to synthesize membranes, ertiesaswellastheoftenhighlyelaboratedstructureofthe
for exine formation and as a source of energy (Pacini pollen wall are species-specific. This is of key importance
1990). Despite the obvious importance of tapetum, it is not only for pollen interaction with papillary cells on the
possible to achieve the proper maturation of functional stigma but also to facilitate its recognition by pollinators.
polleninvitrofromuninuclearmicrosporesinthepresence Insect-andotheranimal-pollinatedspeciesshedpollenthat
of essential nutrients (Tupy´ et al. 1991). can be decorated with extremely complex surface struc-
The highly specialised biological role of angiosperm tures facilitating its adhesion to the pollinators whereas in
pollen is reflected by the unique composition of the sur- wind-pollinated species these structures are often absent
rounding cell wall. The sculptured pollen wall not only (Fellenberg and Vogt 2015).
protects the male gametophyte and its precious cargo but Polarised microspores undergo an asymmetric division
facilitatesthebroadcommunicationwiththestigmasurface during pollen mitosis I (PMI, Fig. 1) resulting in the for-
(Scottetal.2004).Itsinnerlayer,intine,isofgametophytic mation of two unequal daughter cells with distinct cell
originwhereastheouterlayer,exine,ismostlysporophytic. fates, a large vegetative cell and a small generative cell.
The synthesis of pollen wall begins already in tetrads, Bothcellsarepresentinthespaceboundbythemicrospore
immediately after the completion of meiosis. Microspores cell wall. The generative cell soon migrates into the veg-
in tetrads first synthesize the pectin-cellulose primexine etative cell to form a unique ‘‘cell-within-a-cell’’ structure
functioning as a matrix for the depositionof sporopollenin (Russell and Jones 2015; Russell et al. 1996). PMI repre-
precursorsprecedingtheirsubsequentpolymerization.Asa sents another critical moment in the male gametophyte
complex compound of fatty acids and phenylpropanoids, development; it ensures the fixation of the ongoing male
sporopollenin belongs among the toughest known gametophytic developmental program, as demonstrated in
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variousplantspeciesbytranscriptomic(Bokvajetal.2015; basedontheobservationthattricellularpollenwasusually
Honys and Twell 2004; Wei et al. 2010) and proteomic quicker in the onset of pollen germination and subsequent
studies (Chaturvedi et al. 2013; Grobei et al. 2009; tube growth (Brewbaker 1967). Furthermore, the basal
Holmes-Davisetal.2005;Ischebecketal.2014;Noiretal. angiosperm Annona cherimola was shown to shed both
2005;Sheoranetal.2006).Thereforethereversalfromthe bicellularandtricellularpollenindifferentratiosaccording
gametophytictothesporophyticdevelopmentisachievable to the surrounding air temperature (Lora et al. 2009). In a
only with unicellular microspores but not with bicellular highertemperature(30 (cid:3)C),almosthalfofthepollengrains
pollen grains (Gaillard et al.1991).PMI alsoresultsinthe were tricellular whereas at lower temperature (20 (cid:3)C), the
initiation of the male germline; the generative cell retains bicellular pollen grains strongly prevailed. It was specu-
its proliferative activity and divides once more during lated that in warmer weather, a higher proportion of tri-
pollen mitosis II (PMII) to produce two male gametes, the cellular pollen was biologically relevant since it allowed
sperm cells (Berger and Twell 2011; Twell 2011). The faster germination and fertilization that was beneficial
disruption of the asymmetry of PMI either by centrifuga- sincestigmatissueswereshorter-livedinawarmerclimate.
tion (Terasaka and Niitsu 1987), by the application of On the other hand, at lower temperature a higher propor-
microtubule-destabilizing agents, e.g. colchicine (Eady tion of bicellular pollen can successfully be more widely
et al. 1995; Zaki and Dickinson 1991), or in particular dispersedduetoitshigherdehydrationandlongerlifespan.
pollenmutants,e.g.MOR1/GEM1(Parketal.1998),leads This effect is further supported by a longer life span of
to the formation of two similar cells with the vegetative pistilsundermildertemperaturesaround20 (cid:3)C(Loraetal.
cell fate. Arabidopsis MOR1/GEM1 (Park et al. 1998; 2009).
Twell et al. 2002) and its tobacco ortholog TMBP200 (Oh
et al. 2010) encode microtubule-associated proteins, plant
orthologs of the MAP215/Dis1 protein family. Phenotype Progamic phase: catch me if you can
defects resulting from the knock-out of the above genes
clearlydemonstratedtheimportanceofmicrotubulesinthe As pollen grainreaches thepapillary cells ofthestigma,it
establishment of cellular polarity preceding PMI, asym- is rehydrated and activated (Firon et al. 2012). Under
metry of which is the key factor in the initiation of male invitroconditions,Arabidopsispollendoesnotincreasein
germline (Twell 2011). volume during the first 21 min of activation (Vogler et al.
The generative cell undergoes yet another mitotic divi- 2015).Theperiodofnoapparentpollengrainenlargement
sion—PMII—resultingintheformationoftwospermcells. is even longer in more dehydrated bicellular pollen
The vegetative nucleus remains in a close physical con- (Barnabas and Fridvalszky 1984). Then, the pollen tube
nection with the two sperm cells after PMII forming the emergesandstartsgrowingwithgraduallyincreasingspeed
male germ unit (MGU). It plays the vital role not only in (Vogler et al. 2015). The pollen tubes growth rate reaches
the delivery of sperm cells to the female gametophyte, but up to 1 cm per hour, placing them among the fastest-
especially in direct communication between the cells and growing plant cells (Lim and Gumpil 1984). The pollen
their nuclei of somatic origin and the germline (McCue tube growth dynamics and its dependence on stored
etal.2011)perhapsincludingthemodulationoftheirgene nutrition reserves differs between studied bicellular and
expression (Slotkin et al. 2009). tricellular pollen species (Mulcahy and Mulcahy 1988).
The PMII can occur before or after pollen maturation Bicellular pollen tube growth consists of two phases—the
and therefore the mature pollen grain can be shed as first growth period is relatively slow with no formation of
bicellular (in which the vegetative cell engulfs a single callose plugs. The second growth period showed more
undivided generative cell) or as tricellular (where the rapid growth characteristics accompanied by callose plugs
vegetative cell is associated with two sperm cells) (Brew- formation (Fig. 2c). Growth during the first period con-
baker 1967). From an evolutionary point of view, the sumed the reserves carried by the pollen grain itself
bicellular pollen is a plesiomorphy trait whereas the tri- whereas the second phase mostly relied on nutrition from
cellular pollen represents an advanced trait. However, it the style. On the other hand, tricellular pollen started to
was postulated recently that a reverse transition from tri- grow quickly together with callose plug formation that
cellular to bicellular pollen has also occurred (Williams reliedonstylarnutritionfromthebeginning(Mulcahyand
et al. 2014). Thus, some species sheding bicellular pollen Mulcahy 1988).
underwent two evolutionary changes rather than keeping Pollentubegrowthisnotisodiametricbuttogetherwith
the original trait permanently (Williams et al. 2014). The root hairs, fungal hyphae and vertebrate axons represents
maturepollengrainhasadehydratedcytoplasm;bicellular an example of tip growth (Palanivelu and Preuss 2000;
pollen was usually shown to be in a more dehydrated and Sˇamaj et al. 2006). It is characterized by continuous
quiescentstatethantricellularpollen.Thisassumptionwas elongation at one tip of the cell without any further
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Fig.2 Pollentubeapical
region.aSchemeofthelily
pollentubetipshowingactin
cytoskeletondynamicsand
pollentubezonation(adapted
fromHeplerandWinship
2015);individualstructuresare
notdrawntoscale).bCa2?and
H?gradientsinlilypollentube
apex(adaptedfromHeplerand
Winship2015);individual
structuresarenotdrawnto
scale).cDistributionofcallose
(arrow)inactivatedtobacco
pollengrainsandduringpollen
tubegerminationobservedby
anilinebluestaining.Callose
plugsareonlyvisible[4hpost
germination(Hafidhetal.
unpublisheddata)
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PlantReprod
divisions of the vegetative cell. Pollen tubes can reach a filaments are randomly oriented so they are organized into
maximum of 50 cm (Mascarenhas 1993) in plant species anetstructure.Thistip-mostregionisshapedasareversed
with long styles, for example maize. Tip growth requires cone and beside the net structure of actin filaments; it
several mechanisms that are evolutionary conserved in typicallycontainsvesicles(HeplerandWinship2015).The
various tip-growing tissues of different organisms: vital importance of this actin net structure in the apical
cytoskeleton organization, vesicular trafficking, small zone was demonstrated by the application of latrunculin B
GTPasessignalling andiongradient formation(Palanivelu thatstronglyimpaired pollen tube growth andmorphology
and Preuss 2000; Sˇamaj et al. 2006). Moreover, novel (Vidali et al. 2001). Latrunculin B binds to the actin
formation of cell wall is required in the growing pollen monomersandblocksthemfrompolymerizationwhilstthe
tubessincetheyusuallyoutgrowthediameterofthepollen stable actin filaments remain unimpaired by latrunculin B
grain several-fold. This is achieved by the formation of treatment. The role of microtubules in the growing pollen
callose plugs in regular distances to maintain constant tubes was thought to be less important and originally it
amountofcytoplasmrequiredforfillingtheinsidespaceof seemed that the organelles and vesicles were mainly
thepollentube(Fergusonetal.1998;Mogamietal.2006). transported along actin filaments since the colchicine
From this point of view, pollen tube contains several application did not stop pollen tube growth (Heslop-Har-
zones. The tip-most zone is called clear zone as it looks rison et al. 1988). However, later, mitochondria were
‘‘clear’’ under the microscope because the organelles pre- reported to be transported along microtubules in pollen
sent there have quite low refractivity—in particular, the tubes grown in vitro (Romagnoli et al. 2007) and tubulin
starch-containingamyloplastsaremissingfromthispartof was shown to be part of RNA storage particles (Honys
pollentube(HeplerandWinship2015;Vidalietal.2001). et al. 2009). Recently, a more prominent role of micro-
This clear zone comprises two distinct regions, apical and tubules in pollen tubes (for instance in vesicular transport)
subapical (Fig. 2a). The apical region is typical for its was reported (Onelli et al. 2015). Microtubules are there-
inverted cone-shape, in which endoplasmic reticulum ele- forelikelytoattractmoreattentioninthefuturepollentube
ments and vesicles are present. On the other hand, the studies.
subapical region of the clear zone contains also Golgi The distributionof pollen tube organelles aswell as the
apparatus and mitochondria. Behind the clear zone, there organization of pollen tube growth rely also on ion fluxes
arealsolargerorganelles,suchasamyloplastsandvacuoles and gradients. Among them, calcium ions (Ca2?) and
(Lancelle and Hepler 1992). The refractivity of this region protons(H?)areofakeyimportance(Michardetal.2009).
isalsohigherthanthatoftheclearzonesoitsappearanceis Ca2? ions show a gradually increasing concentration
quite different in both light and electron microscopes towards the pollen tube tip, a distribution usually called
(Hepler and Winship 2015). ‘‘tip-focused gradient’’ (Fig. 2b). In the pollen tube apex,
Fast pollen tube growth and overall morphology is the Ca2? concentration reaches 1–10 lM concentration,
underlaid by the organization and function of the ten- to hundredfold higher than in the pollen tube shank
cytoskeleton. Starting behind the subapical zone of lily with Ca2? concentration ranging between 0.1 and 0.3 lM
pollen, actin filaments are organized in parallel bundles (Holdaway-Clarke et al. 1997). An increased Ca2? con-
(Staiger et al. 2010; Wilsen et al. 2006). The cortical fila- centration in the pollen tube apex can serve as a signal for
mentstransportvesiclestowardsthetipwhereasthecentral Ca2?/calmodulin protein kinases, some of which were
filamentsarededicatedforthebasipetalvesiculartransport identified in pollen trancriptome and/or proteome (Sup-
(Fig. 2a). Thus, the vesicular flow resembles a reversed plementary Table 1 and the section below). The Ca2?
fountain. Myosins, the motor molecules along the actin gradient is also crucial for the regulation of actin
filaments,movetowardsthebarbedendsofmicrofilaments. cytoskeleton dynamics since Ca2? ions activate villin/gel-
In order to allow the transport in the desired direction, the solin,whichpromotesthedestabilizationofactinfilaments
corticalandcentralfilamentsareorientedtheoppositeway: (RenandXiang2007).Moreover,theactivityofprofilinin
the central ones have their barbed ends facing the tube tip monomer actin sequestration is promoted by Ca2? (Kovar
whereas the cortical filaments face the tip by their pointed et al. 2000). Thus, these two proteins collectively desta-
ends (Lenartowska and Michalska 2008). Near the border bilize actin filaments and prevent its re-polymerization in
between pollen tube apical and subapical regions an actin thetip-mostregionofthepollentube.Itshouldbestressed
collarisformedanditspositionislikelytoberegulatedby once morethattheincreasedCa2?concentrationisonly at
a higher pH 7.5 and Ca2? gradient. In comparison, pH at the pollen tube tipsince further fromthe tip, theexcessive
the very tip of the pollen tube reaches 6.5 (Feijo´ et al. Ca2? ions are sequestered by endoplasmic reticulum and
2004). These two factors (pH and Ca2? concentration) mitochondria and the Ca2? concentration is kept at the
control actin polymerization together with actin-binding normal physiological values (reviewed in Hepler and
proteins activity (see below). In the apical region, actin Winship 2015). Another process that is promoted by an
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PlantReprod
elevated Ca2? concentration is exocytosis (Camacho and application of brefeldin A that acts as a GNOM inhibitor
Malho 2003). (ZhangandMcCormick2010).Rabproteinsareassociated
Theprotongradientisformedinasimilarway—protons with endomembranes and show usual specificity to par-
enterthepollentubecytoplasmviaprotonpumpsatthetip ticular parts of the endomembrane system (Woollard and
wherethereisaslightlyacidicpH(pH6.5).Thedistalband Moore 2008). RAB11B in tobacco and RABA4D in Ara-
in the clear zone remains, on the other hand, slightly bidopsis were shown to be required for a correct pollen
alkaline(pH7.5;Feijo´ etal.2004)(Fig. 2b).Interestingly, tubegrowth(deGraafetal.2005;SzumlanskiandNielsen
thisapicalacidicpHarearesemblesthereverse-coneshape 2009). Not surprisingly, these small GTPases were
discussed above (Michard et al. 2008). Similar to calcium important for pollen tube growth since they are vital for
ions gradient, the pH variation contributes to the actin exocytosis of various compounds such as cell wall pre-
cytoskeleton dynamics via actin-binding proteins. The cursors, membrane components and signalling molecules
slightly alkaline pH in the subapical region promotes which promote pollen tube growth (Qin and Yang 2011).
ADF/cofilin complex that cuts the actin filaments to more Rop GTPaseswith bound GTP are usually localized in the
parts(Allwoodetal.2002;Chenetal.2002;Lovy-Wheeler apical plasma membrane. They execute several important
et al. 2006; Staiger et al. 2010) and thus promotes actin functions such as organization of the actin cytoskeleton.
dynamicsinthecorticalregionofpollentube.Intheshank They generate reactive oxygen species, and mediate cal-
andatthepollentubetip,thereisneutralpH7.0orslightly cium-dependent signaling (Zheng and Yang 2000; Sˇamaj
acidic pH 6.5, respectively, under which ADF/cofilin is etal.2006).ROP1wasdiscoveredinapicalmembranesof
inactive. tobaccoandlilypollentubes(Fuetal.2001;ZhaoandRen
Taken together, the ion fluxes and gradients are not 2006). Not only the GTPases themselves but also their
static in a growing pollen tube but exhibit regular oscilla- regulating proteins such as RhoGDI were shown to play
tions (Feijo´ et al. 1999; Holdaway-Clarke et al. 1997; Shi important roles in pollen tube growth (Klahre et al. 2006);
et al. 2009). These gradient oscillations together with they are integrated into the cellular signalling network of
cytoskeleton dynamics are reflected in pollen tube growth. kinases acting both upstream and downstream the Rop
For example, growth rate of in vitro cultivated lily pollen GTPases (Feher and Lajko 2015).
tubes oscillates between 0.1 and 0.4 lm s-1 within
15–50 s (Pierson et al. 1996). All of the above mentioned
factors seem to be coordinated but a general pacemaker is Reserves storage and mobilization: shop till you
still not known (Michard et al. 2009). drop
Both cytoskeleton dynamics and ion gradient oscilla-
tions tightly co-operate with the activity of small GTPases Thevegetativecelloftheimmaturepollengraincontainsa
from the Rab, Arf and Rop/Rac families that play a key dense cytoplasm with numerous organelles. The maturing
role during vesicular transport and other processes in the pollen grainshowsconsiderablemetabolicactivityandthe
growing pollen tube (Sˇamaj et al. 2006). These small vegetative cell accumulates a considerable amount of
G-proteins have two states, the active form binding GTP various metabolic reserves including carbohydrates, pro-
and the inactive form bound to GDP (Bishop and Hall teinsandlipidsnecessaryfortherapidgrowthofthepollen
2000). Moreover, the activity of the small GTPases is tube(Pacini1996;Pacinietal.2006).Aspecificportionof
regulatedbythreemainclassesofproteinfactors—guanine reserves comprises osmoprotectants, e.g. disaccharides,
nucleotide exchange factors (GEFs), GAFs and GDIs. proline and glycinebetaine, protecting cellular membranes
GEFs promote the activity of small GTPases by exchang- and proteins from damage caused by dehydration
ing GDP for GTP. The GTPases inactivate themselves by (Schwacke et al. 1999). The generative cell inherits from
their GTPase activity, and this inactivation can be accel- the microspore a very small portion of the cytoplasm and
eratedbyGTPaseacceleratingfactors(GAFs)thatpromote organelles.Whereasthegenerativenucleuscontainshighly
the GTPase activity of G-proteins. Finally, guanine condensed chromatin, the larger vegetative nucleus with
nucleotide dissociation inhibitors (GDIs) conserve the numerous pores exits the cell cycle in G1 phase and con-
G-protein in the GDP-bound form and thus block GTPase tains decondensed chromatin. Therefore higher transcrip-
reactivation (Feher and Lajko 2015). tional activity of the vegetative nucleus in comparison to
Arf GTPases are involved inthe vesicular transportand the generative cell can be assumed; however, that is not
localizetobothendosomesandGolgiapparatus.Mutations negligible either (Borges et al. 2008).
directly affecting Arf GTPases themselves were not From PMI to the maturity, pollen accumulates both
described in pollen tube functional studies. However, mRNAandproteins(Hafidhetal.2011;Honysetal.2009).
several experiments showed their roles during pollen tube In this period, pollen volume doubles, the amount of total
growth by analysing gnom (Arf GEF) mutants or by RNA increases seven times and mRNA content increases
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PlantReprod
thirteen to twenty times (Schrauwen et al. 1990; Tupy´ Protein glycosylation is a co-translational or post-
1982). Pioneering experiments using transcription and translational covalent attachment of carbohydrate chains
translation inhibitors in tobacco pollen tubes showed that (glycans) to the polypeptide backbone. According to the
transcription was detected only during the first several atom, bywhich thecarbohydrateresiduesareboundtothe
hours of the pollen tube growth, which led to the conclu- peptide, three types of glycosylation are recognized. N-
sion that pollen tube growth was mainly dependent on glycosylatedproteinsbeartheglycansonnitrogenatomsof
translation but virtually independent of transcription asparaginintheAsn-X-Ser/Thrconsensussequence,where
(Cˇapkova´ et al. 1988). However, the introduction of high- X is any amino acid except proline, serine, and threonine
throughput technologies led to the identification of (Lerouge et al. 1998). O-glycosylation is executed on the
numerous genes transcribed specifically after pollen ger- oxygen atom of serine, threonine or hydroxyproline
mination in vitro in all species studied—Arabidopsis (Qin (Hanisch 2001). Finally, rare S-glycosylated proteins carry
et al. 2009; Wang et al. 2008), rice (Wei et al. 2010) and their glycan moiety on the sulphur atom of cysteine
tobacco (Hafidh et al. 2012a, b). Moreover, many tran- (Stepper et al. 2011).
scriptsweredenovosynthesizedeveninthetobaccopollen Protein glycosylation usually increases protein stability
tubes cultivated as long as 24 h (Hafidh et al. 2012a). andplaysaroleinprotein–proteininteractions(Uedaetal.
Interestingly, the interaction of the pollen tube with the 1996). Likewise, many pollen allergens are glycosylated
pistil tissues, during which pollen tubes gained the com- (reviewed by Puc 2003). Glycoproteins or more heavily
petence for fertilization (Higashiyama et al. 1998; Palani- glycosylated proteoglycans are important components of
velu and Preuss 2006), activated a specific set of 1254 cell walls and they are often found in association with
genes that were not detected in in vitro cultivated pollen membranes or as secreted proteins. In all these compart-
tubes(Qinetal.2009).Moreover,383ofthesegeneswere ments,numerousmembersofalargefamilyoftheproline/
pollen-enriched. De novo expression of genes involved hydroxyproline-rich glycoproteins are prominent. This
predominantly in signal transduction, transcription and family was originally classified into three separate classes:
pollen tube growth in pistil-activated pollen tubes sug- none or subtly glycosylated proline-rich proteins (PRPs),
gested the possibility of a female-responsive regulatory moderately glycosylated extensins and heavily glycosy-
network orchestrating pollen tube gene expression upon latedarabinogalactan-proteins(AGPs)(forreviewseeEllis
growth through the pistil (Qin et al. 2009). A set of pistil- et al. 2010; Wu et al. 2001). The latter two classes are
activatedgenesrequiredforpollentubedifferentiationand defined by the presence of O-glycosylated hydroxyproline
sperm cells release was later found to be controlled by as hydroxyproline-rich glycoproteins (HRGPs) and their
threeSIVpollen-tubeexpressedrelatedMYBtranscription heterogeneity and abundance attributed them to multiple
factors—MYB97, MYB101 and MYB120 (Leydon et al. functions in plant growth and development, plant defence
2013). and signalling (Ellis et al. 2010). Recently, HRGPs were
In tobacco, stored transcripts were shown to be associ- identified as a component of calcium signalling pathway
ated with large translationally silent ribonucleoprotein (Lamport and Varnai 2013).
particles (EPP complexes; Honys et al. 2000). EPP com- InArabidopsis,aspecific subsetofAGPswasshownto
plexes are associated with the cytoskeleton and contain be expressed in reproductive tissues with the majority of
small and large ribosomal subunits (Honys et al. 2009). them being present in female tissues along the path taken
Upon pollen activation and during subsequent pollen tube bythe growingpollen tube (Pereira et al.2014).However,
growth,thetranscriptsstoredinEPPsarede-repressedand four AGPs, namely two classical AGPs (AGP6, AGP11)
translated (Honys et al. 2009). Furthermore, EPP particles and two AG-peptides (AGP23 and AGP40) were specifi-
are likely to be transported towards the tip of the growing cally expressed in pollen and pollen tubes (Nguema-Ona
pollen tube and could represent a transport form of the et al. 2012). A subset of AGPs, namely those expressed in
transcripts originating from mature pollen. The translation the male gametophyte, are attached to the plasma mem-
itself leads to production of native polypeptides that brane by glycosylphosphatidylinositol (GPI) anchor
undergo a plethora of possible post-translational modifi- (Lalanne et al. 2004). One function of these proteoglycans
cations (PTMs) to form a functional mature protein prod- is the control of nexine formation (Jia et al. 2014) and
uct. These include phosphorylation, methylation, subsequent pollen germination. Higher number of early
glycosylation, myristoylation, acetylation etc. (Knorre germinating pollen tubes within the anthers was observed
etal.2009)thatareusuallyessentialfortheproperprotein in agl6/agl11 double and agl6/agl11/agl40 triple mutants
structureandfunction.Herewewillfocusinmoredetailon (Nguema-Ona et al. 2012). Accordingly, the knockout of
protein glycosylation and phosphorylation since these pollen-expressed AGPs resulted inareduced seed set. The
modificationshavebeenstudiedmostintenselyinthemale expressionpatternofmalegametophyticAGPsissupposed
gametophyte. to be balanced since the up-regulation of AGP40 and
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AGP23 was observed in agl6/agl11 double null mutant 2012).Someoftheseprotocolsdealwiththechallengesof
pollen tubes (Nguema-Ona et al. 2012). Recently it was protein extraction and phosphoprotein/phosphopeptide
reported that the expression of all four pollen-specific enrichment from very tough tissues such as mature pollen
AGPs is directly controlled by the AT-hook nuclear grain (F´ıla et al. 2011, 2012; Mayank et al. 2012; Sheoran
localized (AHL) family DNA-binding TEK protein (Jia et al. 2009). Many phosphoproteomics studies have been
et al. 2014), which therefore indirectly controls nexine performedinplants(Lietal.2015;Meyer etal.2012;van
formation in the pollen wall (Lou et al. 2014). Bentem et al. 2008; Wolschin and Weckwerth 2005),
Extensins(EXTs),thesecondlargeclassofHRGPs,are however, none of these studies focused on pollen
alsopresentinthecellwallwheretheyareinvolved inthe phosphoproteome.
formationofcrosslinkingnetworks.Thereforetheiractivity The first male gametophytic phosphoproteomics study
is predominantly observed in fast growing cells like root identified 962 phosphopeptides corresponding to 598
hairs and pollen tubes (Cannon et al. 2008; Lamport et al. phosphoproteins in Arabidopsis mature pollen (Mayank
2011). Unlike AGPs, extensins do not show strict pollen- et al. 2012). Notably, a high number of phosphoproteins
specific expression patterns and their co-expression in (240 in particular) were newly identified. The most
pollentubesandroothairswasobserved(Dupl’a´kova´ etal. prevalent phosphoprotein categories were regulation of
2007;Hruzetal.2008).ThisisalsothecaseforEXT18,a metabolism and protein function, signal transduction and
classical extensin required for vegetative growth, repro- cellular transport. Many kinases were identified, implying
ductive development, pollen viability and fertility. Among that kinases themselves were phosphorylated, for instance
other phenotypic defects, ext18 mutants show significantly AGC protein kinases, calcium-dependent protein kinases,
slower pollen tube growth and reduced seed set (Choud- and SNF1-related protein kinases.
hary et al. 2015). Intobacco,theproteinphosphorylationdynamicsduring
Althoughprominent,O-glycosylatedHRGPsarenotthe pollen activation was studied (F´ıla et al. 2012). There, dry
only glycoproteins associated with male gametophyte mature pollen grains and pollen suspension activated
function.Intobaccopollentubes,twohighlyabundantcell in vitro for 30 min were compared. In total, 139 phos-
wall N-glycoproteins of 66 and 69 kDa were identified phoprotein candidates carrying 52 phosphorylation sites
(Cˇapkova´ et al. 1997). The block of their N-glycosylation were identified (F´ıla et al. 2012). Most phosphoprotein
by tunicamycin caused reduction of the callose deposition candidates were associated with energy metabolism, a
intopollentubecellwallandconsequentlyimpairedpollen category that has to be precisely regulated after the pollen
tubegrowth.Glycoproteinsofsimilarsizeswerefoundalso tube hydration. Other meaningful overrepresented protein
inseveralotherangiospermspecies(Fidlerova´ etal.2001). categories were protein destination and storage, metabo-
In spite of the abundance and apparent important function lism, cell structure and protein synthesis. Several phos-
of these N-glycoproteins, their exact molecular activity is phopeptides were found to be shared by both Arabidopsis
still unknown. Moreover, protein N-glycosylation was and tobacco pollen (F´ıla et al. 2014) pointing to the com-
demonstrated to be of vital importance in pollen tube per- mon nature of pollen activation in angiosperms. Pollen
ception(Lindneretal.2015).TheEVANandTURANgenes phosphoproteomicsstudiesalsosignificantlycontributedto
encode putative uridine diphosphate (UDP)-glycosyltrans- the public PhosPhAt database of Arabidopsis thaliana
ferase superfamily protein and dolichol kinase, respec- phosphorylationsites(Dureketal.2010;Heazlewoodetal.
tively. It is likely that proteins responsible for pollen tube 2008). Many newly identified phosphorylated proteins
perceptioninfemaleorgansarestronglyglycosylatedsince were likely to be pollen-specific or -enriched as demon-
mutations of these genes taking part in N-glycosylation strated by their transcription profiles (Dupl’a´kova´ et al.
pathwaycausedprematurepollentubeburst(Lindneretal. 2007; Hruz et al. 2008; Mayank et al. 2012).
2015). Therefore membrane- and cell wall-associated gly- Interestingly, changes of the phosphoproteome in a
coproteins are not only important for pollen tube growth gymnosperm Picea wilsonii pollen and pollen tubes were
through the pistil but are also good candidates to mediate studied in response to nutrient depletion from pollen tube
male–female cross-talk during double fertilization. cultivation media (Chen et al. 2012). 42 phosphoproteins
Protein phosphorylation is rather fast, dynamic and were found to be differentially regulated. Of them, phos-
transientPTM,anditsreversiblenaturepredestinesitfora phorylation of proteins involved in cytoskeleton dynamics
regulatoryrole.Theswitch from ametabolicallyquiescent was found to be specifically responsive to Ca2? and
pollen grain to a rapidly growing pollen tube has to be sucrose deficiency (Chen et al. 2012). These three studies
precisely regulated by several mechanisms including pro- applied different phosphoproteomic approaches, and thus
tein phosphorylation. In the past decade, plant phospho- thedatasetsbetweenthemarenotcomparablesinceevery
proteomics evolved rapidly and nowadays, several protocol biases towards a different segment of phospho-
protocols are available (Dunn et al. 2010; F´ıla and Honys proteome (Bodenmiller et al. 2007). However, together
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they provided interesting input in the regulatory processes involves both mechanical/physical orientation and che-
in male gametophyte. motropic guidance of the pollen tube by the female
Protein phosphorylation can be studied directly by reproductivetissues.Thephysicalguidanceisattributedto
phosphoproteomic approaches as mentioned above or theorganizationoftransmittingtracttissues(TT)aswellas
alternatively, from the perspective of protein kinases its secreted compounds. Some of the identified secreted
(Supplementary Table 1). Several kinase motifs were compounds include sulfinylated azadecalin (S-azadecalin;
reported amongst the phosphorylation sites both in Ara- Qin et al. 2011), c-aminobutyric acid (GABA; Ling et al.
bidopsis and tobacco male gametophytes (F´ıla et al. 2012; 2013; Palanivelu et al. 2003; Yu et al. 2014), brassinos-
Mayank et al. 2012) and few of them were shared by both teroids(Vogleretal.2014),aswellasotherhormonesand
species (F´ıla et al. 2014). However, this in silico approach metabolites that direct pollen tube growth towards the
only revealed the presence of individual motifs but the ovule. Whereas the chemotropic guidance is associated
actual link between a particular protein kinase and target with secreted signals by the attractant (the ovules) either
proteins is still missing. To study the protein kinases pre-laid along the pollen tube path towards micropylar
activity themselves, several pollen-specific kinases were entry or intensively secreted as diffusible signals ensuring
studied including mitogen-activated protein kinases (MAP successful ovule targeting by the pollen tube (Heslop-
kinases). MAP kinases represent a large family of Ser/Thr Harrison 1987; Heslop-Harrison and Heslop-Harrison
protein kinases common for all eukaryotes (Kultz 1998). 1986; Mascarenhas and Machlis 1962). The majority of
They mediate the prolyl-directed phosphorylation on these signals constitute small secreted peptides predomi-
xxxxxxS*Pxxxxx, and xxxxxxT*Pxxxxx peptide motifs nantlyofthedefensin-likecysteinerichsubfamily(DEFL)
(Leeetal.2011).However,asubsetofthesemotifsisalso secreted from the egg apparatus (reviewed by Bleckmann
recognized by cyclin-dependent protein kinases. Different et al. 2014; Higashiyama 2015). Additionally, fail-safe
MAP kinases play their roles in various phases of tobacco mechanisms exist whereby undegenerated female synergid
male gametophyte development. MAP kinase NTF4 was cell persist to attract additional pollen tube in a case of
activatedafterpollenhydrationbutbeforetheactualpollen failed fertilization by the first pollen tube and ensure
tube emerged. Its role is likely in the activation of pollen double fertilization of the female gametes (Kasahara et al.
metabolism(Wilsonetal.1997).InArabidopsis,fourMAP 2012).
kinases were identified in the pollen shotgun proteome This chapter will discuss (1) secreted peptides by the
dataset—MPK6,MPK8,MPK9,andMPK15(Grobeietal. female reproductive tissues with role in pollen tube
2009). Two of these (MPK8, and MPK15) were shown to attraction,(2)factorsofthepollentubethatcouldperceive
bephosphorylatedattheTDYmotif(Mayanketal.2012). female guidance signals directly or indirectly, (3) a brief
However, their exact roles during male gametophyte discussion and transcriptomic analysis of the Arabidopsis
development are not yet clear. The other motif over-rep- DEFL subfamily, cysteine-rich receptor protein kinases
resentedintheArabidopsisphosphoproteomicdatasetwas (CRKs) and GPI-anchored proteins in Arabidopsis as
a basophilic motif RxxS*xx, that is recognized by CaM- potential factors likely to be involved in ovular attraction,
dependent protein kinase family (Lee et al. 2011). In pol- signal perception and pollen tube guidance. Recent com-
len, three calmodulin protein kinases were identified prehensive reviews on pollen tube guidance are available,
(Honys and Twell 2003),oneofwhich was present alsoin see Bleckmann et al. (2014) and Higashiyama (2015).
the pollen proteome and phosphoproteome (Grobei et al.
2009; Mayank et al. 2012). The kinases can also be func- Ovular secreted peptides for pollen tube attraction
tionallystudiedbutthisisintricateduetothecomplexityof
proteinkinases.However,adoublehomozygousmutantof After successful pollination and penetration through the
two AGC protein kinases showed defects in pollen tube stigma, a compatible pollen tube grows through the
growth and their competitiveness but not in a full pene- extracellular matrix of the transmitting tract tissues with
trance (Zhang et al. 2009); however, the connecting link theaidoffemaleguidancesignalstoreachandfertilizethe
with the signaling pathways is still missing. female gametes (Maheshwari 1950; Yadegari and Drews
2004). This cell–cell communication has emerged as an
importantbottleneckforunfavourablefertilizationandasa
Pollen tube guidance: show me the way pre-zygotic barrier for interspecies hybridization. Tech-
niquesinvolvingtheuseofsingle-celllaserablation,useof
As the pollen tube grows through the pistil tissues, it is genetic mutants and high-throughput genomic approaches
guidedtowardsovulestoensuredeliveryoftwonon-motile such as tissue-specific transcriptomic studies have identi-
sperm cells for double fertilization. The guidance process fied various transmittingtract and ovular secreted peptides
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