special paper calcium function and distribution during fertilization in angiosperms

15 Pages·2007·0.27 MB·English

AmericanJournalofBotany94(6):1046–1060.2007.
SPECIAL PAPER
C
ALCIUM FUNCTION AND DISTRIBUTION DURING FERTILIZATION
1
IN ANGIOSPERMS
LI LI GE,2 HUI QIAO TIAN,2,4 AND SCOTT D. RUSSELL3
2SchoolofLifeScience,XiamenUniversity,Xiamen361005,P.R.ofChina;and3DepartmentofBotanyandMicrobiology,
UniversityofOklahoma,Norman,Oklahoma73019USA
Calciumhasanessentialsignaling,physiological,andregulatoryroleduringsexualreproductioninfloweringplants;elevation
ofcalciumamountsisanaccuratepredictorofplantfertility.Calciumispresentinthreeforms:(1)covalentlyboundcalcium,(2)
loosely bound calcium typically associated with fixed and mobile anions (ionic bonding); and (3) cytosolic free calcium–an
importantsecondarymessengerincellsignaling.Pollenoftenrequirescalciumforgermination.Pollentubeelongationtypically
relies on external calcium stores in the pistil. Calcium establishes polarity of the pollen tube and forms a basis for pulsatory
growth.Applyingcalciumonthetipmayaltertheaxis;thuscalciummayhavearoleindeterminingthedirectionalityoftube
elongation.Intheovaryandovule,anabundanceofcalciumsignalsreceptivity,providesessentialmineralnutrition,andguides
thepollentubeinsomeplants.Calciumpatternsintheembryosacalsocorrespondtosynergidreceptivity,reflectingprogrammed
cell death in one synergid cell that triggers degeneration and prepares this cell to receive the pollen tube. Male gametes are
releasedinthesynergid,andfusionofthegametesrequirescalcium,accordingtoinvitrofertilizationstudies.Fusionofplant
gametesinvitrotriggerscalciumoscillationsevidentinboththezygoteandprimaryendospermduringdoublefertilizationthatare
similartothoseinanimals.
Keywords: angiospermsexualreproduction;calcium;cellsignaling;doublefertilization;gameteinteractions;programmed
celldeath.
When compatible pollen lands on the stigma, pollen grains increasinglybeguntoinvestigatefertilizationmechanismsona
germinate, forming tubes, which typically elongate through a biochemical, cell biological, and molecular basis. The role of
pollen aperture. The commonly haploid pollen tube continues calcium in this process has received particular attention, as
to elongate within the sporophytic (commonly diploid) tissue calcium appears to provide a universal signal with pleiotropic
of the pistil, proceeding through the style and, once within the effects involving attraction, long and short-distance communi-
ovary,ontotheplacenta.Thetubeultimatelyreachesanovule cation,cellularfusion,andcellsignaling.Techniquesallowing
andentersthehaploidfemalegametophyte(commonly known theisolation andmaintenanceofpollenandotherreproductive
as the embryo sac). The male gametes are released into the cells have been slow to develop because angiosperm gametes
embryosac,aretransportedtoanappropriatelocation,andthen areinaccessibleanddifficulttomanipulate.Studiesoncalcium
fuse with their respective female target cell where nuclear metabolism in these cells may help us to answer questions
fusion and fertilization occurs. In this process, complex about plant fertility and fertilization. This review is among the
interactions occur between the male gametic cells, female first to examine the role of calcium in sexual reproduction in
gametic cells, and the pistil, permitting attraction and precise floweringplants—atopicthathasnotbeenseparatelyreviewed
positioning of the pollen tube in the style, appropriate ovule in prior reviews on plant calcium biology.
targeting,andreleaseofmalegametes(commonlycalledsperm Calcium is a necessary ion in plant development, with
cells) inside the embryo sac. Release of the male gametes myriad physiological functions. Calcium is tightly compart-
occurs at a precise location within the embryo sac, facilitating mentalizedinspecificstatesofavailabilitywithinplantsandis
gamete translocation and placing sperm cells in a unique requiredfornormalgrowthanddevelopment.Initsionicform,
positionforgameterecognition.Twomalegametesarepresent it is known to interact directly and indirectly with calmodulin
within each pollen tube, and each male gamete fuses with a toregulateotherproteinsthroughsignaltransductionpathways
single female target cell. One male gamete fuses with the egg as a second messenger and to serve as a metabolic factor or
cell, forming the zygote and subsequent embryo lineage, cofactor in various cellular activities. Calcium mediates cell
whereas the other male gamete fuses with the female polar physiological processes through multiple independent path-
nucleiwithinthecentralcell,formingthenutritiveendosperm. ways. For many years, calcium regulation in plant physiolog-
Inthepast30yr,numerousstudiesonpollentubeelongation ical processes has been an active topic of research, and results
and pollen-pistil interactions have been published. Recent have often been reviewed; some general reviews include Bush
research into sexual reproduction of flowering plants has (1993, 1995), Gilroy et al. (1993), and Sander et al. (1999).
Literature reviews summarizing calcium signaling during
pollen tube elongation reflect the particularly active research
1Manuscriptreceived11October2006;revisionaccepted8May2007.
currently proceeding in this area (Hepler, 1997; Taylor and
TheauthorsthanktheU.S.DepartmentofAgriculture(NRICGPgrantno.
Hepler,1997;ZhangandCass,1997;Malho´,1998a;Trewavas
99-37304-8097)andtheNationalNaturalScienceFoundationofChina(grant
no.30670126)forpartialsupportoftheresearchreportedinthisreview. and Malho´, 1998; Yang, 1998; Franklin-Tong, 1999a, b;
4Author for correspondence (e-mail: [email protected]); phone: Robinson and Messerli, 2002; McClure and Franklin-Tong,
þ86-592-2186486;fax:þ86-592-2181015 2006). Among examinationsof calciuminvolvement insexual
1046
June 2007] GE ET AL.—CALCIUM IN FERTILIZATION PROCESS OF ANGIOSPERMS 1047
Fig.1. Transmissionelectronmicrographsofcalciumantimonateandcorrespondingenergy-dispersiveX-rayspectrafromtobaccoovule.Asterisks
indicateosmiophilicregions.(a)Nucelluscellcontainingnumeroussmallcalciumantimonateprecipitates,particularlyincellwall(cw).Bar¼0.2lm.(b)
Calciumantimonateprecipitatesincentralcellvacuoleoftobacco.Bar¼0.2lm.(c)Energy-dispersiveX-rayspectrumcomposedofoverlappingpeaksof
calciumandantimonyincentralcellprecipitates.(d)GaussianmodeloftheprominentLa-andLb-peaksofantimony(Sb)overlappingwiththeKa-peak
of calcium (Ca); lower lines depict contributions of Sband Ca in the composite precipitate spectrum. Reproduced from Tian and Russell (1997) with
permissionofSpringer-Verlag.
plant reproduction, however, the study of calcium interactions ionized form, Ca2þ. Stored calcium is often sequestered in the
in the pistil has been relatively neglected. This current review cell wall, localized in specific cellular organelles, or is
reportsontheroleofcalciumduringsexualplantreproduction, associated with proteins that use calcium as a coenzyme or
including:(1)recentresearchregardingthefunctionofcalcium that control calcium concentration. When physiological
in pollen tubes and pollen tube guidance; (2) spatial and conditions in the cell alter the concentration of Ca2þ, the
temporal characteristics of calcium localization in pistils; and solubilitycoefficient(K )predictsthehighestconcentrationof
sp
(3) general aspects of calcium function during gametic ionic free calcium that can accumulate before it spontaneously
interactions and fertilization in angiosperms. The role of beginsformingionicbonds.IfCa2þexceedstheK foragiven
sp
calcium in early plant development is discussed elsewhere calcium-binding molecule, then excess calcium is bound;
(Anil and Rao, 2001). however, if Ca2þ falls below the K , Ca2þ is dynamically
sp
released from its ionically bound state. Thus, high concentra-
tions of free Ca2þcan be dynamically depleted above the K
sp
TYPES OF CALCIUM IN CELLS by reversible ionic binding, effectively controlling the free
calcium concentration. The effective K of the binding
sp
Calciumincellsexistsinseveralstates.Thetotalcalciumof molecule,whichisoftenaprotein,maybedramaticallyaltered
a cell is generally purported to range between 0.1 and 10 mM in binding and functionality by changes in molecular or
(Nagaietal.,1975;SpanswickandWilliams,1965)andcanbe ionization state, including those induced by pH and other
divided into free calcium, bound calcium, and stored (loosely- environmental factors. Protein alterations through enzymatic
bound)calcium.Freecalciumislocatedinthecytosol,isfreely modification, or linkage of control molecules, including
soluble, and is present at concentrations of about 200 nM phosphorylation, also control calcium binding. Any such
(Brinleyetal.,1977;Bush,1995;Sandersetal.,1999).Bound changes in ionic state of the molecule may alter its ability to
calcium has a stronger affinity with macromolecules, which bindcalcium.Becausetheyoccurinanunboundsolubleform,
often means that it forms covalent chemical bonds requiring some of these calcium binding proteins can serve as transport
substantialenergytobreak.Suchtightlyboundcalciumcannot molecules, acquiring calcium from storage and releasing it
beprecipitatedbycompetitionwithlowaffinityanions,suchas where calcium is required (Tandler et al., 1970).
antimonate. This tightly bound calcium can be detected by a Eachoftheseformsofcalciumhasadifferentphysiological
wide range of non-specific methods, including X-ray analysis function. Changes in free calcium concentrations above a low
(Chandler and Battersby, 1976; Van Iren et al., 1979) and thresholdamountallowfreeCa2þtoactasasecondmessenger,
proton-inducedX-rayemission(PIXE,Reissetal.,1983)(Fig. serving as an early product in signal transduction. Because
1). The highest concentration of bound calcium is found in released free Ca2þ may trigger additional Ca2þ pools to be
common crystalline forms, such as calcium oxalate. Loosely- released through activation of ion channels or conversion of
bound calciumis theprevalent form of calcium stored in most boundtofreecalcium,thisphaseofcellsignalingmayresultin
cells. This form of calcium is typically present at concentra- dramatic amplification of local Ca2þlevels. Such free calcium
tionsabove10(cid:1)6Mandisindynamicequilibriumwiththefree release to the cytoplasm and locally amplified calcium
1048 AMERICAN JOURNAL OF BOTANY [Vol. 94
concentrations characterize cell signaling and transmission of and Herth, 1978). One hypothesis for calcium uptake is that
external stimuli. Receptors like calmodulin and other calcium- pollen hydration may trigger increased turgor that allows
bindingproteinsmaytriggerchangesinactivesitesoralterthe uptakethroughstretch-activatedcalciumchannels(Feijo´ etal.,
activity of ion channels, which may produce spikes in free 1995).Thiscould,inturn,maintaincalciumpolarizationinthe
calcium concentration. Examples of such interactions abound pollengrain,amplifypolarizationofcalciumdistributioninthe
incellbiologyandincludecheckpointreleaseduringmitosisor pollen tube, and create a sustainable gradient.
meiosis.Boundcalciumalsooftenformsanintegralpartofthe During pollen germination, a calcium effect has also been
structureofthecell,particularlyinsuchdensecrystallineforms confirmed using calcium antagonists to inhibit germination
ascalciumcrystals andcell walls. Interestingly, it may also be (Bednarska,1989b), although pollengrainsinsomeplantscan
an early predictor of future programmed cell death, as for germinate in media without calcium. Presumably, the latter
instance in the anther ring cells of tobacco, which fuse the plantshavesomecalciumstoresinthepollenwall,whichmay
loculesofanthersacs(Sandersetal.,2005).Inthetobaccoring bereleasedintothemediumduringpollenhydration(Shivanna
cells, programmed cell death is preceded by the accumulation andJohri,1985; Heslop-Harrison,1987). Inricepollengrains,
of abundant fine druses in vacuoles, which may contribute to abundant calcium exists within the pollen grain before
triggering cell death. germination (Tian et al., 1998).
Dynamically sequestered calcium, as mentioned before, is
typically ionically rather than covalently bound. This calcium
storeisdynamicandmaythereforebereadilyconvertedto(and CALCIUM EFFECTS ON POLLEN TUBE ELONGATION
from) free calcium through interaction with a wide variety of
control proteins—in this sense such proteins have calmodulin- Exogenous calcium in the immediate environment affects
like properties. Presumably, a wider examination of expressed bothpollengerminationandtubegrowth.Afamousassaydone
transcripts and proteomic data in pollen (Hruba et al., 2005; by Mascarenhas and Machlis (1962) revealed a chemotropic
Sheoran et al., 2006) and other tissues (Swanson et al., 2005) response of pollen tubes of Antirrhinum majus to external
may aid in identifying specific protein candidates for such calcium gradients in vitro. In cultured pollen tubes of
calcium binding; information about such products is likely to Tradescantia virginana, the optimum concentration for
be readily available for bioinformatic data mining in interna- elongation was from 10(cid:1)4 to 10(cid:1)3 mol/L calcium in the
tional genome and proteome related databases. To avoid the medium (Picton and Steer, 1983). In other plants, pollen tubes
potentiallytoxiceffectsoffreeCa2þ,theconcentrationofCa2þ grewpoorlyifcalciumconcentrationsinthemediumcontained
in the cytoplasm and nucleus in resting cells is three to four higher or lower concentrations (Morre´ and Vanderwoude,
lower than inother cellular compartments (Bush, 1993; Gilroy 1974; Reiss and Herth, 1980; Steer and Steer, 1989;
et al., 1993). However, when the cell is stimulated by external Obermeyer and Weisenseel, 1991). In culturing pollinated
stimuli, free calcium is released, calcium channels open, and styles of tobacco, pollen tube growth also required supple-
free Ca2þ enters through the plasma membrane. Sequestered mental calcium in the medium (Tian and Russell, 1997b).
calcium is also released from the ER, vacuole, and mitochon- Whentheexternalcalciumconcentrationexceeds5310(cid:1)6M,
dria (Marme´, 1989; Trewavas and Malho´, 1998). Wick and actinfilamentsinthepollentubeofLiliumlongiflorumbecome
Hepler (1982) characterized this non-covalently sequestered fragmented and cytoplasmic streaming stops (Kohno and
calcium as loosely bound calcium because of its exchangeable Shimmen, 1987, 1988). In media containing low concentra-
property. tionsofcalcium,generallythepollentubeapexswells,andthe
cell wall at the tip becomes very thin and discontinuous. In
mediacontaininghighconcentrationsofcalcium,however,the
CALCIUM EFFECTS ON POLLEN GERMINATION wallatthetipofthetubebecomesoverlythickened.Lowering
calcium concentrations appears to diminish the elongation of
When pollen grains are germinated in small numbers in the tube by causing an excessive quantity of vesicles to
vitro, the rates of germination and elongation are often less accumulate at the tip, inducing apical swelling (Morre´ and
thaninlargerscalepollenculture.Thispopulationeffectcanbe Vanderwoude, 1974; Picton and Steer, 1983; Steer and Steer,
overcomebyaddingmorepollen,butitmayalsobeovercome 1989). High calcium concentrations appear to accelerate
by adding more calcium to the medium (Kwack and Brew- vesicle fusion at the pollen tube tip, but cytoskeletal elements
baker, 1961). Confirmation of this phenomenon has been maybealtered,contributingtoathickenedwallatthetubetip.
obtained in 86 species representing 39 plant families (Brew- Externaleffectsofcalciumonpollentubegrowthhavebeen
baker and Kwack, 1963), so this represents a widespread confirmed by adding calcium channel blockers or antagonists
calcium dependency among flowering plants. into the medium (Bednarska, 1989b; Heslop-Harrison and
Uptake of calcium by germinating pollen grains has been Heslop-Harrison, 1990). Pollen tube growth is inhibited by
directly demonstrated using the radioisotope 45Ca2þ (Bed- experimentally opening calcium channels using the calcium
narska, 1989b), which is rapidly incorporated into pollen. ionophoreA23187inthemedium.IonophoreA23187disturbs
Accumulation of calcium in the membranes of germinating the calcium gradients inside and outside the pollen tube,
pollen is readily illustrated using the fluorescence microscopic permitting Ca2þ to pass freely through the plasma membrane
probe,chlorotetracycline(CTC)(Polito,1983).Attheonsetof (Reiss and Herth, 1978, 1979b; Picton and Steer, 1983;
pear-pollen hydration, the first faint CTC fluorescence was Bednarska, 1989a).
located at the periphery of the pollen grain, associated with
germinal apertures. Following hydration, fluorescence contin- Calciumdistributioninpollentubes—Intracellularcalcium
ued to intensify near the pore. Calcium accumulation was also has been measured using a variety of localization techniques.
demonstrated in the apertures of lily pollen using similar Eachmethoddisplaysuniquesensitivitiesandweaknesses,and
techniques, reflecting a distinct polarization of calcium (Reiss the method by which the probes are applied vary in their
June 2007] GE ET AL.—CALCIUM IN FERTILIZATION PROCESS OF ANGIOSPERMS 1049
invasiveness and reliability. As different techniques have
emerged,theyhavebeenappliedtoaccumulatecomplementary
data on the different forms of calcium. Using the radioisotope
45Ca2þ and radioautography, Jaffe et al. (1975) demonstrated
thatexternallyabsorbedcalciumfirstaccumulatedatthepollen
tube apex. Proton-induced X-ray emission (PIXE), which
measures total calcium, indicated that concentrations in pollen
tubes of Lilium longiflorum range from 1300 ppm calcium at
the tip to 800 ppm at the base (Reiss et al., 1983).
Chlorotetracycline (CTC) labeling reveals primarily mem-
brane-boundcalciumandindicates thatmembrane-sequestered
calcium displays a similar calcium gradient (Reiss and Herth,
1978, 1979a, 1985; Picton and Steer, 1983; Polito, 1983;
Bednarska, 1989a). According to ultrastructural examination,
this membrane-bound calcium in the pollen tube is mainly
localizedintheendoplasmicreticulum(ReissandMcConchie,
1988; Cresti and Tiezzi, 1990).
Thedetectionoffreecalciumisthemostdifficult,giventhe
mobility of unbound calcium and its ability to dramatically
change the cytoplasm at exceedingly low functional concen-
trations. In animals, transient spatial and temporal changes in
the quantity of free calcium ions (Ca2þ) are still considered to
be among the first detectable events in a signal transduction
cascade arising from stimulation of the outside of the cell Fig. 2. Pseudocolor ratio imaging of elongating Lilium longiflorum
(Tang et al., 2000; Sanders et al., 2002). In that case, the pollentubesloadedwithfura-2-dextrandisplaysconspicuousoscillations
bindingofligandandreceptorevokesaninternalreleaseoffree of apical [Ca2þ]; elapsed time in seconds is superimposed on
calcium that serves as a potent second messenger triggering correspondingimages.Graph(below)depictspollentubeelongationrate
numerous downstream effects. In pollen tubes of Lilium (in white) vs. simultaneously measured Ca2þconcentration at the tip (in
red).ReproducedfromHoldaway-Clarkeetal.(1997)withpermissionof
longiflorum, concentrations of free calcium estimated using
P.K.Hepler(UniversityofMassachusetts,Amherst,MA,USA).
quin-2fluorescencearereportedas10(cid:1)7mol/Lnearthetipand
2310(cid:1)8 mol/L at the base of the tube (Nobiling and Reiss,
1987). Free Ca2þ concentrations measured using the fura-2 gradients in the pollen tube tip closely correspond with
fluorescenceindicatorwereabout10timeslower,withcalcium vigorous tip elongation (Rathore et al., 1991; Obermeyer and
concentrations of 1.7 to 2.6 lmol/L at the tip and 60 to 100 Weisenseel,1991;Milleretal.,1992;Lietal.,1994;Malho´ et
nmol/L at 100 lm behind the tip (Obermeyer and Weisenseel, al., 1994, 1995; Pierson et al., 1994; Feijo´ et al., 1995).
1991). Fura-2 dextran, which is a membrane- impermeable
injected label, provided Ca2þ measurements that were nearly Oscillation of the calcium gradient and pulsatory growth
one-third lower at the tube tip (490 nmol/L at the extreme tip)
of the pollen tube—Pierson et al. (1994) used more rapid,
and 170 nmol/L at 10–20 lm behind the tip. The free-calcium
ratiometric calculations with a confocal microscope and
gradientreportedly dropssharplywithin10–20lmofthetube
confirmed the steepness and depth of the tip-focused
tip (Miller et al., 1992). Although values of free calcium in
intracellular Ca2þ gradient of lily pollen tubes, which varied
these reports differ markedly, a free-calcium gradient appears
from.3.0lMattheapexto;0.2lMwithin20lmfromthe
consistently regardless of technique. Several more recent
tip. Although levels of intracellular Ca2þ were assumed to
reports have indicated that the Ca2þ gradient is restricted to
remain essentially stable over time, further examination
just the apical 20 lm of the pollen tube tip. Changes in free
revealed that, based on measurements using a Ca2þ-specific
calcium beyond the first 20 lm of the pollen tube were
vibrating electrode, extracellular calcium entered the pollen
significantly lower (Pierson et al., 1994, 1996; Malho´ and
tube at influx rates between 1.4 and 14 pmol(cid:2)cm(cid:1)2 s(cid:1)1. These
Trewavas, 1996; Franklin-Tong et al., 1997; Hepler, 1997;
rates of extracellular calcium influx into the pollen tube were
Holdaway-Clarke et al., 1997; Messerli and Robinson, 1997).
uneven but reasonably periodic. The tubes elongated in pulses
The most modern approach uses the recombinant yellow
that corresponded with periodic deposition of cell wall
cameleon calcium reporter. This technique avoids the loading
components (Pierson et al., 1995). Ratiometric ion imaging
problems of the former free calcium probes and is temporally
sensitive enough to detect fluctuations in the calcium gradient of the intracellular Ca2þgradient indicated that the high point
ofthepollentubetip.Also,itsproteinreportercanbetargeted ofthegradientfluctuatedinmagnitudefrom0.75tomorethan
using specific peptide additions to selected subcellular 3 lM during ;60-s measuring intervals. Elevation of the
compartments (Watahiki et al., 2004). calcium gradient appeared to be correlated with an increased
Interestingly,theCa2þgradientexistsonlyingrowingpollen rate of tube growth (Pierson et al., 1996). Holdaway-Clarke et
tubes. Upon death or the addition of calcium channel blockers al. (1997) confirmed that the tip-focused Ca2þ gradient
orantagonistsintothemedium,thecalciumgradientdisappears oscillates with the same periodicity as pollen tube growth,
from the tip of the pollen tube, and the tube stops elongating. but learned that the pulses are slightly out of phase. The
When such arrested tubes are transferred to a medium without extracellularcalciuminfluxisdelayedbyabout11scompared
calciumblockersorantagonists,theyrapidlyreestablishaCa2þ with the oscillation of the Ca2þ gradient (Fig. 2). They
gradient and reinitiate elongation. Strongly concentrated Ca2þ therefore inferred that the delay in the extracellular inward
1050 AMERICAN JOURNAL OF BOTANY [Vol. 94
currentmayberelatedtothecyclicdepletionofCa2þstoresin calcium, their spatial localization, and temporal characteristics
the pollen tube immediately before a similarly out-of-phase compound the complexity of calcium mediation during pollen
cycle of replenishment. tube elongation (Hepler, 1997; Malho´, 1998a, b; Yang, 1998;
Determining how Ca2þ oscillations, tube pulsatory growth, Franklin-Tong, 1999a). The following sections summarize
and extracellular calcium influxes are related represents a some significant advances in our understanding of calcium
significant recent advance in understanding the extremely regulation in the context of pollen tube elongation.
dynamicnatureofcalciuminpollentubeelongation(Malho´ et
al., 1998; Trewavas, 1999). Pulsatory tube elongation, how- Cytosolic free calcium function during pollen tube
ever, is not required, even in plants that characteristically elongation—The ability of the pollen tube to reorient tip
display pulsatory pollen tube behavior. Geitmann and Cresti growth is at the heart of understanding how tubes can so
(1998) found that inorganic Ca2þchannel inhibitors La3þand precisely target a single cell within the female embryo sac.
Gd3þcould trigger the pulsating pollen tubes to abandon their Hypotheses that pollen tubes grow toward the ovule by such
rhythm and continue to elongate with steady rates. Organic tropistic mechanisms as chemo-, thigmo- or electrotropism
inhibitors of Ca2þchannels, nifedipine and verapamil, slowed have met with at best equivocal success. What molecular
pulsefrequencybutdidnotblockpulses.Theseresultsstrongly changes would precede and trigger pollen tube curvature?
suggest that at least two types of Ca2þ channels occur in the Calcium gradients have long been implicated in regulating
plasma membrane of the tube tip. If apical elongation of the pollen tube tip elongation, but their role in determining the
pollen tube is inhibited, periodic membrane traffic may still orientation of tube growth was not well supported by
occurwithnearlythesameperiodicityaswasexhibitedduring experimental evidence until the 1990s.
normal tube elongation (Parton et al., 2003). Rui Malho´’sgroup provided evidence that thedirectionality
Pulsatory exocytosis and cyclic Ca2þ correspond well with of Agapanthus umbellatus pollen tubes could be modified by
pulsatorypatternsofpollentubeelongation,butitisbecoming iontophoretic introduction of calcium and by weak electrical
increasingly evident that models of oscillatory pollen tube fields, in which the pollen tubes elongate toward the cathode.
elongation will not be explained only by cyclic cell wall IntroducingalocalizedgradientofionophoreA23187,whichis
looseningunderturgor(MesserliandRobinson,2003).Cellular believed to open calcium ion channels, causes the pollen tube
oscillations apparently involve an integration of plasma tip to reorient toward the A23187. When the Ca2þ channel
membrane ion fluxes that constitutes a ‘‘cytosolic choreogra- blockerGdCl wasaddedtothegrowthmedium,tipelongation
3
phy’’ofprotonandcalciumfluxesthatoperatesinconcertwith became oriented away from GdCl . The region of highest
3
intake fluxes of anions, including potassium and chloride concentration of cytosolic free calcium was closely aligned
(Feijo´ et al., 2001; Zonia et al., 2001). Chloride efflux at the with tip reorientation and the direction of future elongation.
pollentubeapexisinturnaffectedbyfluxesininositol3,4,5,6- A further demonstration of the effect of free calcium was
tetrakisphosphate with momentary interruptions of chloride obtainedbymicroinjectingcagedCa2þintolivingpollentubes.
efflux associated with elongation maxima (Zonia et al., 2002). These injected pollen tubes were then eccentrically irradiated
Pollen tube behavior is also influenced by other ions, notably with UV light near their tips, causing photolysis of the cages
Kþ, which appears to be controlled by gated channels (Fan et andreleaseoffreeCa2þwithinthepollentubeatthatlocation.
al., 2001; Mouline et al., 2002). Increasing information is AresultingtransientriseinfreeCa2þinducedareorientationof
available concerning the involvement of Rop GTPase-depen- tip elongation toward the site of irradiation. Tip growth
dent regulatory proteins (Fu et al., 2001; Baxter-Burrell et al., resumed near the irradiated region and caused a sustained
2002; Gu et al., 2003), Rop1ps (Zhao and Ren, 2006), Rop2 reorientation of tip elongation. The site of tip reorientation
GTPases(Jonesetal.,2002),andRac-likeGTPases(Cheunget corresponded closely with the local release of Ca2þ. This
al., 2003) in the control of endo- and exocytosis in the pollen pattern was reinforced by a decline in Ca2þ levels on the
tube apex (Camacho and Malho´, 2003). opposite side of the tube, completing the reorientation (Malho´
Dynamic calcium concentrations play a central role in etal.,1994,1995; Malho´ andTrewavas,1996). Thus,areasof
increasingly complicated models of growth oscillations in richCa2þwithinagradientcanreorienttipgrowthandthereby
pollen tubes, both in a direct physiological role and more establish the directionality of future pollen tube elongation. A
importantly in signaling (Holdaway-Clarke et al., 2003). relatedstudydemonstratedthatakinaseinthepollentubeapex
Fluctuations in the pollen tube tip-focused calcium gradient may also be involved in regulating localized Ca2þ channel
arenotreflectedinnuclearcalciumlevelsaccordingtoanalysis activity (Moutinho et al., 1998).
using the recombinant yellow cameleon calcium reporter The signal-transduction intermediate inositol 1,4,5-trisphos-
(Watahiki et al., 2004). Clearly, the occurrence of calcium phate(InsP )canreportedlytriggercalciumreleasefromstores
3
oscillationsinpollentubeshasbecomeanimportantpartofour in the cell, as well as serve as a second messenger controlling
current model for understanding calcium oscillation in higher numerous cellular processes (Berridge, 1993). To examine the
plant systems (see reviews by Evans et al., 2001; Gradmann, effect of locally introduced InsP on pollen tube elongation,
3
2001). Malho´ (1998c) microinjected caged InsP into pollen tubes of
3
Agapanthus umbellatus, then photolysed the cages with spot
irradiation. Releasing caged InsP in the apex, subapical, and
3
ROLE OF THE FUNCTIONAL CLASSES OF CALCIUM nuclear regions of pollen tubes resulted in a transient increase
IN POLLEN TUBE ELONGATION ofCa2þintheseregions.WhencagedInsP wasreleasedinthe
3
nuclearorsubapicalregion,thegrowthaxisofpollentubewas
The three types of calcium in plant cells—bound, loosely realigned. An undesirable consequence was that photolysis
bound, and free calcium—although interchangeable, clearly elicited transitory increases in tube elongation, causing tips to
havedifferentfunctionsandusedifferentcontrollingmolecules burst.Theseresultsclearlyindicatethatcalciumisauniqueand
during pollen tube elongation. These multiple forms of separate part of the signal transduction pathway controlling
June 2007] GE ET AL.—CALCIUM IN FERTILIZATION PROCESS OF ANGIOSPERMS 1051
tube guidance. The addition of cAMP, which affects the plasmamembranematerialforexpansion(Hepleretal.,1994).
localized release of Ca2þ, may reorient tip elongation as well These areas of elevated Ca2þ in lily pollen tubes closely
(Moutinho et al., 2001). resembleconditionsinothersitesofintenseexocytosis(Royet
al., 1999).
Calciuminpollentubewall—Amongtheprimarytargetsof Maintenance of the calcium gradient in the tip is
exogenously provided 45Ca2þ is the pollen tube wall (Brew- characteristic of vigorous pollen tube elongation, but mecha-
baker and Kwack, 1963), where the radioisotope incorporates nistic details arestillnotclear.Calciumconcentrations outside
into carboxyl groups in the pectic components of the cell wall the pollen need to exceed those at the tip, suggesting that
(Kwack, 1967). Calcium associated with pollen tube walls is tightly localized calcium channel activity may be sufficient to
frequently abundant but rarely is incorporated directly at the sustain an exceedingly tight gradient. Even a cursory
tip, because calcium-crosslinked pectin may behave as a rigid examination of elongating pollen tubes reveals intense
and insoluble gel (McNeil et al., 1984). The bipartite pollen cytoplasmic streaming in the tube, but there is a transition in
tube wall consists of an inner sheath of callose and an outer this pattern of movement from directional to saltatory
regionoffibrillarlayerscontainingabundantpectin.Thetipof movement toward the tip (Heslop-Harrison et al., 1991). High
the pollen tube is composed of a single pectin layer (Heslop- concentrations of calcium at the tip induce fragmentation of
Harrison, 1987). In immunocytochemical studies, at least two actin filaments and inhibit cytoplasmic streaming at the
types of pectin were labelled in the pollen tube wall: a extreme tip because of F-actin instability (Kohno and Shim-
polygalacturonic acid form of pectin and methyl-esterified
men, 1987, 1988). The high concentration of calcium within
pectin. Acid pectins capable ofcrosslinking Ca2þarelocalized
thefinal20lmofcytoplasmatthetubetipappearstocreatea
in the subapical region of the pollen tube. In contrast, richly
relatively calm environment for vesicle transport and fusion
methyl-esterifiedpectinsareparticularlyabundantatthepollen
(Steer and Steer, 1989). The negatively charged vesicles
tube tip, where they may inhibit Ca2þ crosslinking. Secretory
(Heslop-Harrisonetal.,1991)maythencontinuetheirforward
vesicles in the pollen cytoplasm also appear to contain
migrationthroughattractiontothepositivelychargedCa2þ-rich
abundant methyl-esterified pectin (Li et al., 1994; Geitmann
tip.Althoughthehighcalciumconcentrationinhibitscytoskel-
et al., 1995; Jauh and Lord, 1996; Geitmann and Parre, 2004).
eton formation in this region, it promotes the directed
Tube elongation represents a delicate balance between the
transportation of secretory vesicles and their adherence and
ability of the tube wall to withstand bursting and the high
fusion with the plasma membrane of the tube tip.
native osmolality of the pollen cytoplasm (Cresti and Tiezzi,
Sperm cells in growing pollen tubes are typically located
1992; Feijo´ etal., 1995). When secretory vesicles release their
somewhat distant from the tip, for instance ;90 lm from the
methyl-esterified pectin at the tube tip, their contents merge to
tip in maize, a location where calcium concentrations are
become incorporated into the tube wall. Upon formation, this
reasonablylow(G.Zhangetal.,1997).Thisconditionseemsto
tipregionisthemostelasticinthetubeandisalsolikelytobe
improve the long-term viability of sperm cells. Maintaining
the expansion point under internal turgor pressure. Following
isolated sperm cells requires a suitable concentration of
tube tip expansion, methyl-esterified pectin migrates basipe-
exogenous calcium for membrane integrity (perhaps ,1
tally from the tip region. Pectin is deesterified and becomes
mM), but the concentration is critical or the useful lifespan
crosslinked with Ca2þ, which contributes much to the rigidity
ofviablespermcellsisverybrief(G.Zhangetal.,1992,1995).
andmechanicalstrengthofthepollenwallaswellasproviding
turgorpressureintheotherregionsofthetube(Lietal.,1994).
In vivo, the region of the pollen tube wall adjacent to the
CALCIUM DISTRIBUTION IN STIGMATIC TISSUE
micropyle in ovules of tobacco and Plumbago contains
abundant calcium-induced precipitates, indicating high con-
When a compatible pollen grain lands on the stigma, it
centrationsoflooselyboundcalcium(TianandRussell,1997a;
germinates and forms a pollen tube with an elongating tip.
Tian et al., 2000).
Usingchlorotetracycline(CTC),TirlapurandShiggaon(1988)
Other calcium functions during pollen tube growth—The found abundant membrane-bound calcium in papillae of
tip gradient of calcium in the living pollen tube is essential to Ipomoea batatas. Bednarska (1989c), who also used CTC,
vesicle secretion at the tube tip. As discussed before, all found a similar result in Ruscus aculeatus L. and confirmed
attempts to alter the gradient also alter the pattern of this with X-ray microanalysis, further observing that germi-
exocytosis. The elevated concentration of calcium near the nating pollen of Primula officinalis and R. aculeatus absorb
secretory membrane appears to be essential for vesicle fusion, calcium from the stigma (Bednarska, 1991). Studies using
as indicated by its involvement in animal as well as plant potassium antimonateprecipitationindicate thatlooselybound
systems (Almers, 1990; Zorec and Tester, 1992; Battey and calciuminsunflowerismoreabundantonthereceptivesurface
Blackbourn, 1993). During membrane fusion, the role of Ca2þ of the stigma, especially outside and inside the papillae, than
appears to be to draw the docked vesicle membrane into on nonreceptive surfaces (J. Zhang et al., 1995). Abundant
appositionwiththeplasmamembranebycounterbalancingthe calcium antimonate precipitates are also reported in the
native negative charge of their constituent phospholipids intercellular matrix of the stigmatic tissue of cotton (J. Zhang
(Battey et al., 1999). Vesicle secretion at the tip of the pollen et al., 1997) and on the stigmatic surface of Brassica oleracea
tube is an intense, localized form of exocytosis that, perhaps following pollination, particularly when the pollen grain lands
not surprisingly, involves an especially steep gradient of and tube germinates on the stigmatic papillae (Elleman and
elevated calcium. In pollen tubes, highly active Golgi bodies Dickinson, 1999). The occurrence of abundant calcium in and
produce abundant vesicles that migrate to their fusion site via onstigmaticsurfacesisinaccordwiththeneedforextracellular
actomyosin associations. The vesicles fuse with the plasma calcium for pollen germination and tube elongation in vitro
membraneatthetipofthepollentube,secretingbothwalland (Brewbaker and Kwack, 1963).
1052 AMERICAN JOURNAL OF BOTANY [Vol. 94
CALCIUM DISTRIBUTION IN STYLAR TISSUE the activity of protein kinases. Ca2þ influx differs between
compatible and self-incompatible plants, suggesting an active
After pollen germination, the tubes penetrate the stigmatic role for the influx of extracellular Ca2þ in the SI response
surface and grow into the transmitting tissue of the style. (Franklin-Tong et al., 2002). In Nicotiana alata, a plant with
Mascarenhas and Machlis (1962) found a chemotropic gametophytic self-incompatibility, self-pollen tube elongation
response of pollen tubes to calcium and observed a gradient was inhibited by an S-related ribonuclease in the stylar
ofcalciumconcentrationsinpistiltissuesofAntirrhinummajus transmitting tissue (McClure et al., 1990). Presumably,
proceeding from the stigma to the ovule. Based on in vitro calcium-dependent protein kinases from the pollen tube
assays, they concluded that the tubes responded to a calcium activatedtheS-ribonucleaseofthetransmittingtissue,resulting
gradientinthepistil,whichcausestubestoelongatetowardthe inthedigestionofRNAinthepollentubeandtheinhibitionof
embryo sac. Calcium gradients have been confirmed in the tube growth (Kunz et al., 1996). Immunological studies have
pistils of a number of flowering plants, including Gladiolus indicated the occurrence of calmodulin and calmodulin-like
gandavensis (Day et al., 1971), but calcium gradients were and calreticulin-like proteins that could be involved in Ca2þ-
absent in several other flowering plants (Glenk et al., 1971; related cell signaling during pollen–pistil interactions (Lenar-
Mascarenhas,1975).Morerecentassaysusingantimonatehave towska et al., 2001, 2002; Rudd and Franklin-Tong, 2001,
confirmed that loosely bound calcium stores occur in the 2003; Snedden and Fromm, 2001).
transmitting tissue of a number of plants, including Brassica
napus(Maoetal.,1992),HelianthusannuusL.(J.Zhangetal.,
1995), Gossypium hirsutum L. (J. Zhang et al., 1997), and CALCIUM DISTRIBUTION IN OVULAR
Lilium longiflorum (Zhao et al., 2004). Calcium-induced SOMATIC TISSUE
antimonateprecipitatesweremainlylocalizedintheapoplastic
systemofthetransmittingtissue,i.e.,intheintercellularmatrix After pollen tubes emerge from the transmitting tissue and
and cell wall, where pollen tubes elongate. In contrast, fewer entertheovary,theyelongateontotheplacentalsurfacewithin
calcium precipitates were localized within the cells of the the ovarian locule. As pollen tubes travel along the placental
parenchymatoustissue.Theoccurrenceofabundantcalciumin surfacetowardtheproximalendoftheovary,individualpollen
the transmitting tissue suggests that the calcium required for tubes diverge from the masses by 908 before elongating
tube elongation in vitro is obtained from the rich calcium through the last several micrometers to enter the ovular
environment of the style during in vivo growth. Calcium as a micropyle. In all plants investigated to date (pearl millet,
directional cue, however, appears to be effective only in the Chaubal and Reger, 1992a; sunflower, J. Zhang et al., 1995;
pollentubesofarestrictednumberofplantsbecausenumerous tobacco,TianandRussell,1997a;cotton,J.Zhangetal.,1997;
flowering plants are insensitive to such a calcium signal Brassica napus, Yu et al., 1998; Plumbago zeylanica, Tian et
(Johnson and Preuss, 2002; Higashiyama et al., 2003). al., 2000; Crocus, Chichiricco´ et al., 2002), the micropyle
Calcium is also implicated in the self-incompatibility appears to contain abundant levels of calcium, which correlate
responseofselfincompatible(SI)plants.WhenS-glycoprotein closely with fertility and may serve as an attractant in some
is extracted from self-incompatible stigmas of Papaver rhoeas plants. Accumulations of calcium-induced precipitates of
andisintroducedintotheculturemedium,theconcentrationof antimonate at the entrance of the micropyle exceeded calcium
calcium increases in tubes, and this increase appears to inhibit concentrations in the integument and nucellus in ovules of
tube elongation (Franklin-Tong et al., 1993). However, when tobacco and Plumbago zeylanica. Calcium distribution ap-
either compatible or heat-denatured incompatible stigmatic S- peared to be polarized within the micropylar nucellus, which
glycoproteinswereintroducedintotheculturemedium,norise contained more calcium-induced precipitates than did the
in Ca2þ levels of pollen tubes was detected, and elongation chalazal nucellus and thus contribute to an apparent calcium
continued normally (Franklin-Tong et al., 1993). Caged Ca2þ distribution gradient (Tian and Russell, 1997a; Tian et al.,
introduced into pollen tubes did not alter tube elongation, but 2000).
upon cage photolysis, a similar inhibition of tube elongation Some spatial and temporal characteristics are evident in the
wasobservedaccompanyingartificiallyelevatedinternallevels distribution of loosely bound calcium in the ovule. Calcium
of Ca2þ. Photoactivation of caged InsP elicits a similar concentration in the micropylar regions of each ovule are
3
inhibitory response (Franklin-Tong et al., 1996). The potential highestasthepollentubeapproachestheovulesintobaccoand
effect of contamination by other stylar components was Plumbago zeylanica. After the tip of the pollen tube passes,
eliminated by cloning the SI-gene and expressing it in E. coli; calciumlevelsquicklydecline.Flowersinwhichpollinationis
the purified gene product also elicited the same result. Thus, withheld, however, retain higher concentrations of micropylar
the S-protein alone appears to be sufficient for triggering the calcium,withcalciumlevelsdecreasingslowlyovertime.That
Ca2þsignalduringthepollenSIresponse(Franklin-Tongetal., the gradient is retained suggests that micropylar calcium
1995). gradients remain. Delayed pollination tests also demonstrate
Imaging the calcium directly established that S-protein that the ovule continues to attract pollen tubes (Tian and
addedtothemediumresultedinaninfluxofextracellularCa2þ Russell, 1997a; Tian et al., 2000). Accumulating calcium
at the ‘‘shank’’ of the pollen tube. The influx of extracellular concentrationsmaybeavaluableindicatoroffertility(Chudzik
Ca2þ appears to play a role in the Papaver SI response and Sniezko, 2003).
(Straatman et al., 2001). The addition of the Ca2þ-antagonist Generally, only one pollen tube enters each ovule in
VerapamilorthecalciumchannelblockerLa3þallowedpollen angiosperms. In ovaries with only a single ovule, subsequent
of Secale to overcome the self-incompatibility response and pollentubesstopgrowingonceonepollentubegrowsintothe
permitted elongation into the style (Wehling et al., 1994). ovule. However, once multiovulated ovaries are penetrated by
Calcium is believed to regulate special transduction reactions pollen tubes, arriving tubes simply bypass the penetrated
during the self-incompatibility response, likely by controlling ovules to grow toward receptive ovules. After the pollen tube
June 2007] GE ET AL.—CALCIUM IN FERTILIZATION PROCESS OF ANGIOSPERMS 1053
penetrates the ovule, rapid decreases in micropylar calcium loosely bound calcium (Tian and Russell, 1997a). The
correlate closely with loss of receptivity and may prevent degenerated synergid of tobacco contains higher levels of
supernumerary pollen tubes from entering the ovule. Presum- calcium than the persistent synergid (Huang and Russell,
ably, the disappearance of micropylar calcium renders 1992). According to Mogensen and Suthar (1978), synergid
penetrated ovules unattractive to the remaining pollen tubes. differences originate during the first day after pollination.
This latent calcium signal, controlled by penetration of the Localization of calcium using potassium pyroantimonate
ovule, can be rapidly dissipated. confirms a correlation between calcium differences between
the two synergids before and during pollen tube penetration
(Tian and Russell, 1997a). The pollen tube penetrates the
CALCIUM DISTRIBUTION IN THE EMBRYO SAC synergid that has the most label, and the pattern of calcium
labeling characteristic of the persistent synergid is retained in
The embryo sac represents the end of the journey for the the unpenetrated synergid after fertilization (Tian and Russell,
pollen tube. Because in most angiosperms the pollen tube 1997a).
enters the receptive synergid, calcium distribution in the Abundanceofcalciuminthesynergidiscloselyrelatedwith
synergids has been the subject of multiple studies conducted pollen tube entrance. Although no differences in calcium
using a variety of calcium detection techniques. The earliest contentareevidentinthetwosynergidsinunpollinatedflowers
examination of the chemical content of synergids was a study ofsunflower, calciumcontent after pollinationincreases inthe
byJensen(1965)inwhichsectionsoftheembryosacofcotton synergidthatdegeneratesandreceivesthepollentube(Heand
were microincinerated and then examined using dark field Yang, 1992). Similarly, calcium content and distribution are
microscopy. Abundant ash, presumed to be essentially similar in the sister synergids of B. napus before pollination,
elemental calcium based on the temperature used, was found butcalciumprecipitatesinbothsynergidsincreaseby2.4times
localized within the synergids. Since this early work, other afterpollination,andadifferenceinsizeofprecipitategranules
techniques have been used to document the abundance of appears to differentiate the two synergids. The synergid that
calciuminthesynergidofotherspecies.Whentotalcalciumin will accept the pollen tube contains smaller granules than the
the embryo sacs of wheat and pearl millet before and after persistentsynergid(Yuetal.,1998).Intobacco,calciumlevels
pollination was assayed using energy-dispersive X-ray analy- in the synergid appear to remain elevated until after a pollen
sis, concentrations greatly exceeded typical physiological tube enters the receptive synergid, when the high level of
measurements;critical-pointdriedsynergidvacuolescontained calciuminthefiliformapparatusappearstodecrease(Tianand
over20%calciumbymass(ChaubalandReger,1990,1992a). Russell,1997a).FollowingpollinationinToreniafournieri,the
CTClabelingofmembrane-boundcalciuminembryosaccells egg apparatus produces a labyrinthine structure on the
of tobacco and Petunia hybrida indicated intense fluorescent micropylar surface that contains high levels of calcium. Once
signal in the receptive synergid (Huang and Russell, 1992; thesynergidispenetratedbythepollentube,however,calcium
Tirlapur et al., 1993). Loosely bound calcium is similarly content decreases in the penetrated synergid (Kristo´f et al.,
abundant, according to localizations using potassium antimo- 1999), and the ovule no longer appears to attract pollen tubes
nate (Chaubal and Reger, 1992a, 1993, 1994; He and Yang, (Higashiyama et al., 1998). In this context, calcium may be
1992; Tian and Russell, 1997a; Yu et al., 1998; Kristo´f et al., only a smallpart of a larger process of programmed cell death
1999).Withpotassiumantimonate,changesincalciumlabeling that occurs in the synergid as part of pollen tube arrival and
in the receptive synergid can be detected before degeneration discharge (Christensen et al., 2002).
andbeforeotherultrastructuralcues(TianandRussell,1997a). A continuing interaction between elongating pollen tubes
Calcium-induced antimonate precipitates also provide an and receptive synergids is evident in terms of calcium
opportunitytomaploosely-boundcalciumbasedonprecipitate signaling. Calcium in the synergid, especially in the filiform
distribution, size, and quantity (Tian et al., 2000; Yu et al., apparatus, directly attracts elongating pollen tubes, whereas
1998). elongatingpollentubesmaystimulatecalciumaccumulationin
In all flowering plants examined in recent years, except the embryo sac (Tian and Russell, 1997a). Similar to calcium
Plumbago zeylanica (which lacks synergids), the highest dynamics in the micropyle, penetration by a pollen tube
calcium levels within the embryo sac occur in synergids— induces rapid calcium depletion in the synergids, which may
and typically, the most abundant label is localized in the prevent extra pollen tubes from entering the embryo sac
receptive synergid. Calcium in the synergid appears to be (Chaubal and Reger, 1990, 1992b).
distributed in a micropylar–chalazal gradient. The highest Once the pollen tube penetrates the embryo sac, calcium in
concentration of calcium appears to occur in the filiform the degenerated synergid may play a role in sperm cell
apparatus in wheat, pearl millet, tobacco, and Brassica napus transport and fusion. In flowering plants, the two sperm cells
(ChaubalandReger,1992a,b,1994;TianandRussell,1997a; andvegetativenucleusformafunctionalassociationknownas
Yu et al., 1998). In sunflower, however, higher concentrations the ‘‘male germ unit’’ (Dumas et al., 1984). This assemblage
of calcium appear to occur at the chalazal rather than the favors the transportation of the male gametes within the tube
micropylar end of the synergid (He and Yang, 1992). In all of and assures their simultaneous delivery, but must be severed
these plants, the embryo sac contains two synergids, only one upon arrival in the embryo sac. Because the assemblage is
of which receives the pollen tube and sperm cells. In mature presumably maintained by cytoskeletal elements (Palevitz and
embryo sacs of wheat, pearl millet, sunflower, and B. napus, Tiezzi, 1992), calcium in the synergid may be involved in the
both sister synergids contain nearly the same calcium content breakdown of cytoskeletal elements from the pollen tube and
(Chaubal and Reger, 1990, 1992a, b; He and Yang, 1992; Yu the preparation of the sperm cell surface for fusion.
etal.,1998).IntobaccoandPetuniahybrida,however,thetwo Among the cells of the embryo sac, the egg cell appears to
synergids containdifferent levelsof membrane-bound calcium retain consistently low levels of calcium (Chaubal and Reger,
(Huang and Russell, 1992; Tirlapur et al., 1993) as well as 1992a,b;TianandRussell,1997a;Yuetal.,1998;Tianetal.,
1054 AMERICAN JOURNAL OF BOTANY [Vol. 94
2000). However, in Plumbago zeylanica, which lacks syner- did not improve fusion success, Faure et al. (1994) concluded
gids, the egg cell in the mature embryo sac contains abundant that 5 mM calcium stimulated but did not coerce fusion. In
calcium, especiallyinits filiform apparatus(Tian etal.,2000). contrast,fusionmaybecoercedathighcalciumconcentrations
This result supports the idea that calcium attracts pollen tubes (50 mM), particularly when accompanied by high pH (Kranz
totheovuleandintotheembryosacinvivo andconfirmsthat and Lo¨rz, 1994). Under these conditions, fusions among a
theeggcellofP.zeylanicahasindeedacquiredsomesynergid- variety of cell types are possible, apparently overriding any
like functions (Cass and Karas, 1974; Russell, 1982). After existing cell recognition systems. Although fusions of isolated
fertilization in rice, calcium-induced antimonate precipitates tobacco sperm and egg cells could be accomplished using
increase by 4.4-fold in the cytoplasm of the zygote and by polyethylene glycol without calcium, the addition of 5 mM
10.5-fold in the nucleoplasm. Calcium levels in two-celled CaCl improved success in preliminary in vitro fertilization
2
proembryos, however, quickly decline in the cytoplasm, but experiments (Tian and Russell, 1997c). Lower levels of
remain abundant in nucleoli and cell walls. An abundance of calcium are typically used accompanying electrofusion (e.g.,
calcium was reported in the apoplast of the embryo sac after 2 mM CaCl in rice; Uchiumi et al., 2006).
2
fertilization and during early embryogenesis (Zhao et al., Calcium alone, however, cannot replace the need to have
2002). gametes at the appropriate position in the cell cycle to initiate
The central cell also contains significant accumulations of successful embryogenesis; this cell phase at which fusion
calcium,particularlyatthemicropylarendofthecell(Tianand normally occurs differs among angiosperms(Friedman, 1999).
Russell, 1997a; Tian et al., 2000; Yang et al., 2002). After the Althoughtobaccocellsreadilyformcallusandplantsareeasily
second sperm cell fuses with the central cell, labeled calcium regenerated from callus, tobacco gametes isolated at G phase
1
also quickly decreases in the newly formed endosperm. There did not successfully form embryos (Tian and Russell, 1997c).
seems to be no direct relationship between calcium in the A detailed examination of the role of the cell cycle in tobacco
central cell and chemotropic activity of pollen tubes in vivo. fertilization showed that sperm cells underwent S phase upon
Calcium gradients near the synergids presumably control the arrivalattheovuleandcompletedSphasewithinthesynergid.
attraction of pollen tubes, whereas calcium in the central cell GameticfusionthenoccurredatthebeginningoftheG phase,
2
mayrelatemorespecificallytothestimulationofgametefusion and completion of S phase in the female gametes was
and the early development of the endosperm, which precedes synchronized with the male gametes (Tian et al., 2005). In
that of the embryo. Because the central cell is a major tobacco, only gametes in which the sperm and egg cells fused
nutritionalstorefortheembryosac,highlevelsofcalciummay atG phaseformedsuccessfulembryos,whereasmalegametes
2
also facilitate the absorption of nutrients into the embryo sac. of tobacco isolated from the pollen tube at G phase formed
1
Abundant calcium has been demonstrated to be necessary for fusion products that failed to divide. Cell cycle congruity is
accumulation and transport of amino acids (Rickauer and thus a prerequisite for successful in vitro fertilization, even if
Tauner, 1986) and for carbohydrate metabolism (Brauer et al., calcium requirements are met (Wang et al., 2006).
1990). In late stages of microspore development in rice, the Ultrastructural studies of embryo sacs have frequently
antherloculealsocontainsabundantcalcium,whichappearsto revealed highly electron dense materials between the egg and
be related to rapid starch accumulation (Tian et al., 1998). centralcellsnearthesiteoffuturegametefusion.Althoughitis
Additionalexperimentalevidencewillbeneededtofurtherour unclear whether these materials are directly involved in fusion
understanding of the role of calcium in the central cell. in vivo (Russell, 1992), with the antimonite technique,
Knowledge on the function of the antipodal cells is very abundant calcium precipitated at the fusion site in tobacco
limited in most plants. In many, the antipodal cells degenerate (Tian and Russell, 1997a), Brassica napus (Yu et al., 1998),
prior to embryo sac maturity and have no obvious function in and Plumbago zeylanica (Tian et al., 2000). Presumably, an
the embryo sac. In these plants, antipodal cells presumably environment containing sufficient calcium favors the fusion of
contain low levels of calcium, which distinguishes them from male and female gametes.
the other cells of the embryo sac (Tian and Russell, 1997a). Spontaneousfusionofspermcellpairs—aconditionthought
Although antipodal cells in Plumbago zeylanica may contain toberareinnature—maybestimulatedinspermcellsinvitro.
some calcium granules, most are localized in the single large Using solutions containing 4.27 mM calcium, Roeckel and
vacuole, with no apparent gradients formed. After fertilization Dumas (1993) found spontaneous sperm cell fusions that
ofthecentralcell,antipodalcellsdegenerateastheendosperm formed large, initially multinucleated cells in isolated maize
begins to develop (Tian et al., 2000). gametes.Undercertaincultureconditions,mediacontainingas
low as 1 mM calcium were able to induce sperm cell fusions
consisting of as many as 20 nuclei (G. Zhang et al., 1997).
CALCIUM AND FUSION OF GAMETES Without calcium in the medium, however, spontaneous fusion
oftobaccospermcellsinfrequentlyoccurredandtook.30min
Calcium is known to induce somatic fusion and appears to tocomplete.Infusionmediacontainingcalcium,thefrequency
contribute to gameticfusion as well. In an in vitro fertilization was markedly increased, with cells fusing in as little as 10 s,
studyinmaize,thehighestfusionsuccesswasobtainedusing5 suggesting a direct stimulation of sperm cell fusion (Tian and
mM Ca2þ in the fusion medium (Faure et al., 1994). Fusion Russell, 1998). The spontaneous ability of sperm cells to fuse
was highly successful in sperm–egg cell pairs (79.8%), among themselves decreases as the cells mature, suggesting
whereas only 1.8% of sperm–mesophyll protoplast combina- maturational changes in the sperm cell surface during
tions fused. Under these circumstances, gametes adhered and development. Within 20 h of sperm cell formation, spontane-
fused within ;1 min. This fusion specificity has been oussperm–spermfusionisnomorefrequentthanatthetimeof
interpreted as indicative of gametic cell recognition (Faure et gamete fusion and requires enzymatic treatment (Tian and
al., 1994). Because lowering or eliminating calcium from the Russell, 1998).
medium inhibitedfusionandraising thecalciumconcentration Because calcium-mediated prefertilization events are possi-
June 2007] GE ET AL.—CALCIUM IN FERTILIZATION PROCESS OF ANGIOSPERMS 1055
ble in angiosperm gametes, Williams et al. (1997a, b)
examined the uptake kinetics of Ca2þ and the effect of
exogenous calcium on protein phosphorylation and the Ca2þ-
bindingproteins,calmodulinandcalreticulin,inspermcellsof
maize (Zea mays) in the presence and absence of Ca2þ.
Calcium uptake kinetics indicated that Ca2þ influx in maize
sperms is mediated by a combination of active (oxidative
phosphorylation-dependent) Ca2þtransporters and Ca2þchan-
nels(Williamsetal.,1997a).Withtheadditionof1mMCa2þ,
thephosphorylationofa18-kDaproteinincreased13-foldafter
12 h incubation, calmodulin content increased by 136% in as
little as 1 h, and calreticulin increased by 34% after 3 h, as
compared to the controls (Williams et al., 1997b).
Calciumconditionsduringinvivofertilizationhavenotbeen
directly examined in the intact egg cell because of its relative
inaccessibility. A calcium requirement, however, seems likely.
Because sperm cellsusuallyenter theembryo by means of the
synergid, the calcium conditions of this region may represent
the normal environment for gamete fusion. If this is the case,
the amount of calcium in the immediate environment of the
synergidmayberemarkablyhigh(Higashiyama,2002),butthe
concentration of calcium actually participating in fusion is
unknown.
ACTIVATION OF THE EGG CELL
AND CALCIUM SIGNALING
In animals, the entry of the sperm into the egg cell triggers
Fig. 3. Pseudocolor ratio imaging of intracellular Ca2þconcentration
embryogenesis, but eggs can also be activated by other
ofZeamayseggcellloadedwithfluo-3acetoxymethylesterafterinvitro
disturbances in the surface of the egg. The most conspicuous
fertilization (concentrations in arbitrary fluorescence units). Numbers
initialstepineggactivationisasignificantincreaseinthelevel
indicatesecondsaftersperm–eggfusion,illustratingtemporalandspatial
of cytosolic free calcium and dramatic oscillations in calcium distributionoftheCa2þtransient.ReproducedfromDigonnetetal.(1997)
influx within the fertilized egg cell (Ridgway et al., 1977; withpermissionofC.Dumas(E´coleNormaleSupe´rieuredeLyon,Lyon,
SwannandOzil,1994;Tangetal.,2000;Sandersetal.,2002). France).
InthebrownalgaFucusserratus,fertilizationoftheeggcellis
accompanied by uptake of calcium from the external medium,
theinfluxofCa2þwasclosetozero.Followinggametefusion,
which appears to be necessary for egg cell activation (Roberts
however, a Ca2þinflux was evident near the site of sperm cell
andBrownlee,1995).Calciumuptakebyitselfcanalsotrigger
egg activation, because the addition of the calcium ionophore entry.TheonsetofCa2þinfluxappearstoprecedetheelevation
A23187totheculturemediumwillcausenearlyhalfoftreated of cytoplasmic Ca2þby approximately 40–120 s. The addition
mouse oocytes to undergo parthenogenesis (Nakasaka et al., of 10 lM Gdþ3 prior to fusion is sufficient to block sperm
2000). incorporation, suggesting that the Ca2þ influx is required for
In flowering plants, changes in membrane-bound Ca2þ in incorporation. The fusion point, however, displays a highly
maize egg cells are detectable using chlorotetracycline. localized sperm-dependent and Gdþ3-insensitive Ca2þ influx
Enhanced labeling occurs at the egg plasma membrane and (Antoine et al., 2001).
around the egg nucleus prior to gamete fusion (Tirlapur et al., The role of flowering plant sperm cells in stimulating egg
1995). Following in vitro fertilization of a single egg, activationmayinvolvechemicalssharedwithanimaleggcells.
membrane-bound Ca2þ labeling with chlorotetracycline in- UsingasolubleproteinfactorofBrassicaspermcells,Lietal.
creases in the cytoplasm and around the nucleus, but the Ca2þ (2001) were able to trigger calcium oscillations in mouse eggs
signal at the plasma membrane decreases until it is not and therefore suggested that the activating protein may be
detectable (Digonnet et al., 1997). found in both plants and animals. Injection of a sperm extract
Dramatic changes in free calcium also occur during in vitro in Torenia central cells triggers a calcium oscillation and an
fertilization of flowering plant gametes. In vitro fertilized egg accumulation of calcium that mimics endosperm activation.
cells of maize displayed significant changes in free Ca2þ Interestingly,thiseffectcanalsobeproducedbytheadditionof
(Digonnet et al., 1997), which are apparently triggered by caged inositol 1,4,5-trisphosphate. Once the cages are
fusion (Fig. 3). Cytosolic free Ca2þ was unchanged during photolysed and the chemical is released, inositol 1,4,5-
gamete positioning and adhesion. Once fusion occurred, trisphosphate appears to mimic the effects of free calcium
however, free Ca2þ increased for several minutes and then oscillation (Han et al., 2002). Stimulation of the female target
decreased. This transient calcium elevation apparently triggers cells by sperm extract suggests that sperm cells contain a
an influx in extracellular free Ca2þ, which is non-invasively trigger for releasing egg stores of Ca2þ that may activate a
detectableusinganextracellularCa2þ-selectivevibratingprobe myriad of Ca2þ channels in both plants and animals. Inositol
(Antoine et al., 2000). On the egg cell surface prior to fusion, 1,4,5-trisphosphate is known in other systems to stimulate the