Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/291420840

Linksbetweenmorphologyandfunctionofthepollenwall:Anexperimentalapproach

ArticleinBotanicalJournaloftheLinneanSociety·April2016

ImpactFactor:2.53·DOI:10.1111/boj.12378

READS

58

7authors,including:

ChristianRaquin

FrenchNationalCentreforScientificResea…

42PUBLICATIONS507CITATIONS

SEEPROFILE

FrançoisBrisset

UniversitéParis-Saclay

112PUBLICATIONS1,123CITATIONS

SEEPROFILE

BeatriceAlbert

UniversitéParis-Sud11

23PUBLICATIONS364CITATIONS

SEEPROFILE

Pierre-HenriGouyon

MuséumNationald'HistoireNaturelle

156PUBLICATIONS4,557CITATIONS

SEEPROFILE

Allin-textreferencesunderlinedinbluearelinkedtopublicationsonResearchGate,

lettingyouaccessandreadthemimmediately.

Availablefrom:AlexisMatamoro-Vidal

Retrievedon:20April2016

Links between morphology and function of the pollenwall: an experimental approach

ALEXIS MATAMORO-VIDAL1,2,*, CHRISTIAN RAQUIN2, FRANC�OIS BRISSET3, H�EL�ENECOLAS1, BENJAMIN IZAC2, B�EATRICE ALBERT2† and PIERRE-HENRI GOUYON1†

1D�epartement Syst�ematique et Evolution, Mus�eum national d’Histoire naturelle, UMR 7205 MNHN-CNRS, Paris, 75005, France2Universit�e Paris Sud-11. Ecologie, Syst�ematique et Evolution, UMR 8079 CNRS-AgroParisTech,Orsay, 91405, France3Universit�e Paris Sud-11. ICMMO, UMR 8182 CNRS, Orsay, 91405, France

Received 17 April 2015; revised 3 December 2015; accepted for publication 18 December 2015

The wall of pollen grains exhibits morphological variation in many features including apertures, ornamentationand thickness, but the function of these characters remains to be clarified. It has been suggested that they areinvolved in the accommodation of volume changes (harmomegathy). To investigate this further, we developed aprotocol that induces a controlled hydration of the pollen without affecting its metabolism and we applied it to sixspecies differing in their pollen wall morphology. The entry of water caused pollen swelling and volume increaseleading to breakage of the wall and/or of the plasma membrane, such that the per cent of intact grains wasnegatively correlated with the level of hydration. Qualitative and quantitative differences were observed betweenthe species. Breakage of the exine was observed only in pollen lacking apertures and with thin exine. Variationin the exine ornamentation and thickness could explain the interspecific differences observed for the rates ofbreakage of the plasma membrane. Our results suggest that pollen wall morphology matters for survival andmaintenance of pollen integrity further to volume increase due to hydration. We propose a rationale for futurestudies that should allow disentangling the contribution of different pollen morphological and physiologicalfeatures to harmomegathy. © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society,2016, 180, 478–490.

ADDITIONAL KEYWORDS: aperture pattern – exine ornamentation – harmomegathy – naturalselection.

INTRODUCTION

Plant male success depends largely on the viabilityof pollen until the pollen tube reaches and fertilizesovules. This makes the understanding of how pollengrains adapt to environmental fluctuations animportant issue for plant evolution. During its lifecycle, a pollen grain can go through several hydra-tion and dehydration phases. When pollen reachesmaturity in the anther, it is transferred from a liq-uid environment to an atmospheric environment. Inmost angiosperm species, pollen dehydrates andremains in this state until it lands on a compatiblestigma. Once it is on the stigma, the pollen rehy-

drates and eventually germinates (Edlund, Swanson& Preuss, 2004). Additional hydration–dehydrationphases can occur before or during the dispersal ofthe grain, depending on the relative humidity of theenvironment and of the time of dispersal (Lisci,Tanda & Pacini, 1994; Pacini, 2000; Franchi et al.,2011).

Many structural, physiological and molecularmechanisms are used by pollen grains to adjust tochanges in water content and to maintain internalstability (Firon, Nepi & Pacini, 2012). The externalwall of the pollen (the exine) is made of a highlyresistant polymer called sporopollenin. Mechanismsallowing changes in the shape and in the volume ofthe wall in order to accommodate the variation inthe volume of the cytoplasm caused by changinghydration are thus necessary to avoid pollen

*Corresponding author. E-mail: alexis.vidal@ijm.fr†These authors contributed equally.

478 © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 478–490

Botanical Journal of the Linnean Society, 2016, 180, 478–490. With 6 figures

breakage. The term harmomegathy was proposed byWodehouse (1935) to qualify such an accommodationprocess, which is necessary to allow a retraction(bending) of the wall during dehydration or anextension (stretching) during hydration. Here, wepresent an experimental approach aimed atstudying in a quantitative manner how pollen mor-phological and physiological characteristics mayhelp the grain to accommodate a volume increaseand avoid pollen breakage further to hydration. Onthe basis of the data obtained from this approachand from published work, we propose a hypotheticalmodel predicting response of the grain to hydrationdepending on its morphological and physiologicalcharacteristics.

Two characteristics of the pollen wall, aperturesites and exine ornamentation, have been suggestedto be involved in volume-change accommodation onthe basis of comparative and theoretical approaches(Wodehouse, 1935; Payne, 1972; Heslop-Harrison,1979b; Muller, 1979; Blackmore & Barnes, 1986;Thanikaimoni, 1986; Scotland, Barnes & Blackmore,1990; Halbritter & Hesse, 2004; Chichiricc�o, 2007;Katifori et al., 2010; Volkova, Severova & Polevova,2013). Apertures are sites on the wall where theexine is thin or absent (Fig. 1A–D). During dehydra-tion of the grain, the membrane of the aperture sitesfolds inward, so that the edges of each aperture aretouching each other (Volkova et al., 2013), closing upthe aperture site. It has been shown by mathemati-cal modelling that the area and the shape of theapertures contribute to harmomegathy by reducingthe necessity of the wall to stretch and bend in orderto accommodate volume-changes (Katifori et al.,2010). However, even if the apertural sites mayaccommodate a part of the volume-changes, someflexibility of the wall is required: several species pro-duce pollen with tiny apertures or totally lackingapertures (Fig. 1E, F), and they are still able toaccommodate volume changes. This additional flexi-bility might be provided by other properties of theexine.

Exine ornamentation is the pattern of the outerwall of the pollen (Fig. 1A0–F0) and is variable. Forexample, some species produce pollen with network-like exine patterns made of wide spaces bordered byridges of exine narrower than these spaces (reticu-late pattern, Fig. 1D0, E0). Other exine patterns aremade of exine units (pilea) that have loose connec-tions with each other (crotonoid pattern, Fig. 1F0).These exine units may move apart from each otherand facilitate volume-changes (Katifori et al., 2010).Their separation has been considered as adaptationto volume change during water uptake (Hesse,1999). There is also appreciable variation in exinethickness. Some pollen grains have an exine that is

uniformly thin. In these pollen grains, the whole sur-face of the wall can be considered as an aperture.This type of pollen is thus referred to as omniapertu-rate (Thanikaimoni et al., 1984). At the otherextreme, there are pollen grains with a uniformlythick exine.

The innermost layer of the pollen wall, the intine,is beneath the exine and borders the surface of thecytoplasm. The intine is composed of pectin and cel-lulose and it is much more capable of stretchingand contraction than the exine (Heslop-Harrison &Heslop-Harrison, 1982). Variability in the thicknessof the intine wall might affect the stretching pro-cess and the efficiency of volume-change accommo-dation.

Differences in aperture number, wall ornamenta-tion, and thickness, are likely to produce differencesin harmomegathic efficiency. We studied whetherpollen morphology may affect the capacity of thegrain to accommodate a volume-increase resultingfrom hydration. For this, we developed a protocolthat allows the hydration of the grains in a dose-dependent manner, without changing metabolism:pollen grains were placed in solutions with differentconcentrations of a non-metabolic sugar, which cre-ates different levels of hydration without affectingthe metabolism of the grains. We scored the ratesof breakage in the exine and in the plasma mem-brane of pollen grains exposed to four levels ofhydration. This was done for pollen grains of sixspecies that differ in their aperture sites, wall orna-mentation and thickness. The percentage of intactpollen grains was found to be negatively correlatedwith the level of hydration. In addition, qualitativeand quantitative differences between the specieswere observed. We summarize in a model how themorphological properties of the pollen wall couldexplain these differences.

MATERIAL AND METHODS

LIVING MATERIAL

Pollen grains of six species were studied (Table 1).Plants were grown in the laboratory greenhouse(Orsay, France) using material obtained from theParc Botanique de Launay (PBL) for the followingspecies: Carica papaya L. (voucher PBL-000536), Irisgermanica L. (voucher PBL-011139), Jatropha inte-gerrima Jacq. (voucher PBL-018013), Nicotiana syl-vestris Speg. & Comes (voucher PBL-019066),Ricinus communis L. (voucher PBL-020382). ForMatthiola tricuspidata (L.) R.Br., living material wasobtained from seeds provided by Royal Botanic Gar-dens, Kew (voucher K 000984541, seed bank acces-sion number 60501).

© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 478–490

POLLEN WALL MORPHOLOGY AND FUNCTION 479

POLLEN MORPHOLOGY

Pollen morphology was observed with an FEGscanning electron microscope using experimental con-ditions chosen to avoid the use of a metallic coatingand deep penetration in the organic sample. Toachieve that, a 1-kV high voltage was used with a lowprobe current, permitting observation without anyfatal charging effect and allowing a detailed samplesurface view. Before observations, pollen grains were

cleaned using an ultrasonic cleaner and critical pointdried with an Emitech K850 critical-point dryerQuorum Technologies Ltd. Lewes, East Sussex, UK.

For each species, exine thickness (here taken asthe thickness of the wall and of the ornamentation)was measured for ten grains from one individual,acetolyzed according to the technique of Erdtman(1969) and photographed under a light microscope.Measurements were made on the section where the

A A′ B B′

C C′ D D′

E E′ F F′

Figure 1. Pollen morphology of the species investigated (scanning electron microscope images). A, A0, Ricinus commu-

nis. Pollen (A) has three apertures (triaperturate). Exine ornamentation (A0) consists of more or less rounded depres-

sions < 1 lm in diameter and the distance between the depressions is equal or greater than their diameter (perforate).

B, B0, Carica papaya. Pollen (B) is triaperturate and exine ornamentation is perforate (B0). C, C0, Nicotiana sylvestris.

Pollen (C) is triaperturate. Ornamentation (C0) is perforate. D, D0, Iris germanica. Pollen (D) has a unique aperture

(monosulcate). Ornamentation (D0) is a network-like pattern made of wide spaces bordered by ridges of exine narrower

than these spaces (reticulate). E, E0, Matthiola tricuspidata. Pollen (E) has no apertures (inaperturate) and exine (E0) isreticulate. F, F0, J. integerrima. Pollen (F) has no visible apertures, but the exine is uniformly thin such that the entire

wall functions like an aperture (omniaperturate). Ornamentation (F0) is made of exine units (pilea), which have loose

connections with each other (crotonoid).

© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 478–490

480 A. MATAMORO-VIDAL ET AL.

diameter of the grain was highest using ImageJ Soft-ware (U.S. National Institutes of Health, Bethesda,Maryland, USA).

EXPOSURE OF POLLEN GRAINS TO DIFFERENT LEVELS OF

HYDRATION

Freshly opened flowers were collected and immedi-ately taken to the laboratory. All the plants weregrown in the same plot of the greenhouse so they wereexposed to similar conditions at the time of flower col-lection. In the laboratory, opened anthers wereremoved and gently rubbed on a cellophane tape(Hutchinson�, Maplewood, MN, USA.), until anappreciable amount of pollen was spread on the tape.The cellophane tape was then placed in a Petri dish,filled beforehand with six rounds of laboratory filterpaper imbibed with the osmotic medium. As the cello-phane tape is permeable, pollen was in contact withthe osmotic medium, and could easily be removed bytaking out the cellophane tape without touchingdirectly the grains. Petri dishes were sealed and incu-bated for 12 h at 28 °C. To stop the reaction, the cello-phane tape containing pollen was placed on a slidewith Alexander’s stain and covered with a cover glass.Alexander’s stain stains the cytoplasm pink and theexine green. For each slide, a number of grains (aver-aging 274 � 36 grains per slide; N = 90 slides) (Sup-porting Information, Table S1) was monitored for cellmembrane and wall disruption using a light micro-scope. One of the three following states was attributedto each grain: intact; disruption of the plasma mem-brane; or breakage of the exine (with unimpairedplasma membrane). Note that in inaperturate pollenthe disruption of the plasma membrane occurredalways through a local breakage of the exine.

For each experiment, a sample of pollen collectedfrom a mix of flowers of an individual was dis-tributed among four Petri dishes, each of which hada different osmotic concentration. To control for theeffects of manipulation, a portion of the sample wasplaced with the cellophane tape directly on a slidewith Alexander’s stain and covered with a coverglass (control media). We studied as many individu-als and replicates as possible given material avail-ability at the time of the experiments. The numberof individuals studied per species and the number ofreplications are given in Table 1.

The osmotic medium was adapted from a mediumused routinely to germinate pollen grains in vitro(Bergamini-Mulcahy & Mulcahy, 1983). This mineralsalt solution contained 1.62 mM H3BO3, 1.27 mM Ca(NO3)2.4H2O, 0.81 mM MgSO4.7H2O. The solutionwas buffered to pH 6 with 0.2 mM KH2PO4 and0.05 mM K2HPO4.3H2O. To avoid bias caused by thephysiological process of pollen tube growth, we didT

able

1.Listof

thesp

eciesinvestigated

with

theirmorpholog

icalch

aracteristics,number

ofindividuals

studied,number

ofreplicates,

and

totalnumber

of

grainsscored

Species

Taxon

omy

Aperture

pattern

Exine

ornamen

tation

Exine

thickness(lm)

Number

ofindividuals

Rep

licates/individual

Grains

scored

Carica

papaya

L.

Eudicot

–Caricaceae

Tripaerturate

Perforate

1.56�

0.14

11

1500

Iris

germanicaL.

Mon

ocot

–Iridaceae

Mon

osulcate

Reticulate

1.69�

0.30

31

4500

Jatrop

haintegerrimaJacq.

Eudicot

–Euphorbiaceae

Omniaperturate

Crotonoid

0.54�

0,06

13

4420

Matthiola

tricusp

idata

(L.)R.Br

Eudicot

–Brassicaceae

Inaperturate

Reticulate

2.38�

0.13

61

7293

Nicotianasylvestris

Speg

.&

Com

es

Eudicots–Solanaceae

Triaperturate

Perforate

1.62�

0.12

11

1500

Ricinuscommunis

L.

Eudicot

–Euphorbiaceae

Triaperturate

Perforate

1.71�

0.19

41

5505

Total

24,718

For

theex

inethickness,

themea

nmadeacross10grainsandthestandard

dev

iation

are

given

.

© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 478–490

POLLEN WALL MORPHOLOGY AND FUNCTION 481

not add any metabolic sugar to the solution, butadded instead mannitol, which is a non-metabolicsugar. Four different osmotic levels (0, 0.20, 0.45 and1.00 mol L�1) were obtained by adding respectively0, 1.09, 2.46 and 5.47 g of mannitol to 3 mL of themineral salt solution, in a final volume of 30 mL.Pollen grains placed in these media were subjectedto hypotonic conditions in this way (the lower theconcentration of mannitol, the higher the level ofhydration to which the pollen was exposed).

STATISTICAL ANALYSES

To avoid bias due to breakage of pollen caused bymanipulations or by Alexander’s stain, the percent-ages of intact pollen grains and of pollen breakagewere, for each experiment, corrected with the ratesof pollen breakage observed in the control media.This correction had only a slight effect on the resultsbecause the percentages of intact pollen in the con-trol experiments (Alexander Solution) were alwayshigh (mean = 95.9%; SD = 4; N = 18).

We tested for differences between species in thepercentage of intact pollen, the percentage of pollenwith disruption of the plasma membrane and thepercentage of pollen with exine breakage using theKruskal–Wallis (KW) test. When this test yields sig-nificant results, then at least one of the samples isdifferent from the other samples. To test for an effectof the aperture structure (also using the KW test),species were classified in four aperture patterns(omniaperturate, triaperturate, inaperturate andmonosulcate; Table 1). In the same way, differencesbetween pollen ornamentations in the percentage ofpollen with disruption of the plasma membrane weretested using the KW test by classifying the species inthree different patterns (perforate, reticulate andcrotonoid; Table 1). The data could not be analysedstatistically for each of the four levels of hydrationseparately because this resulted in low sample sizes.Thus, the analyses were performed with the datapooled for all the levels of hydration, such that wecould test for differences between species. Neverthe-less, the plots of the raw data for each level of hydra-tion are shown in order to provide global trends.

Tests for correlations between the percentage ofintact pollen and the hydration level and betweenthe percentage of pollen with disruption of theplasma membrane and exine thickness were per-formed using a Kendall test. Non-parametric statisti-cal tests were used as the assumptions of theparametric methods do not apply to our data (theresiduals were not normally distributed). All theanalyses and graphs were performed using R Soft-ware (R Development Core Team, 2010).

RESULTS

DESCRIPTION OF POLLEN MORPHOLOGY

Pollen morphology of the species under investigationis shown on Figure 1 and Table 1. The pollen wall ofRicinus communis, Carica papaya and Nicotiana syl-vestris has three apertures (triaperturate pollen).Pollen of Iris germanica has a single broad aperture(monosulcate). Jatropha integerrima and Matthiolatricuspidata produce pollen with no visible apertures(inaperturate).

Three different exine ornamentations wereobserved. Ricinus communis, N. sylvestris, andC. papaya have pollen with a perforate pattern con-sisting of more-or-less rounded depressions lackingexine (Punt et al., 2006). These depressions are< 1 lm in diameter and the distance between thedepressions is equal or greater than their diameter.Some areas of the pollen of N. sylvestris have elon-gated elements > 1 lm long arranged in an irregularpattern (rugulate pattern), but the dominant patternwas perforate. For I. germanica and M. tricuspidata,the exine exhibits a reticulate pattern (Punt et al.,2006) consisting of spaces > 1 lm bordered by ridges(muri) of exine narrower than these spaces. Theexine surface of J. integerrima has a crotonoid pat-tern. This pattern is like a reticulate pattern exceptthat the network is composed of separated exine ele-ments (pila) instead of muri (Punt et al., 2006).

The results of the measurements of exine thick-ness are given in Table 1. The six species may bearranged into three classes according to their exinethickness. The first class contains J. integerrimawith a relatively thin exine, averaging 0.54 lm. Car-ica papaya, N. sylvestris, R. communis and I. ger-manica form the second class as the mean of theexine thickness of these species ranges between 1.56and 1.71 lm. Matthiola tricuspidata forms the thirdclass, with a relatively thick exine, averaging2.38 lm.

POLLEN HYDRATION RESULTS IN BREAKAGE OF THE PLASMA

MEMBRANE AND/OR OF THE EXINE IN A DOSE-DEPENDENT

MANNER

The percentage of intact pollen increases with theconcentration of mannitol (i.e. with the diminution ofhydration levels) (Fig. 2A). The two variables aresignificantly correlated (Kendall test; s = 0.473;P < 0.001). When looking at each species separately(Fig. 2B), the same trend was observed, but the cor-relation could not be tested because of low samplesizes. Nevertheless, the data suggest that for all thespecies, the lowest performance occurs for thehigh levels of hydration (i.e. for low mannitol concen-trations).

© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 478–490

482 A. MATAMORO-VIDAL ET AL.

Exposure to hydration induced breakage of theplasma membrane in all species studied, and break-age of the exine only in species producing pollenlacking apertures. For the species producing pollenwith apertures (R. communis, C. papaya, N. sylve-stris and I. germanica), only two kinds of pollen wereobserved following hydration: intact pollen and pol-len with a breakage of the plasma membrane(Fig. 3A–G). The disruption of the plasma membraneoccurs through the apertures (Fig. 3B, D, F, G).Breakage of the exine was never seen in these fourspecies. In the species producing pollen lacking aper-tures (J. integerrima and Matthiola tricuspidata),the exposure of pollen to hydration resulted in threedifferent effects on the pollen (Fig. 3H–L): some

grains remained intact (Fig. 3H, J), whereas otherhad only their exine broken (Fig. 3K), and othershad both their plasma membrane and exine broken(Fig. 3I, L). In the last case, the disruption of theplasma membrane occurred through a local breakageof the exine.

PERCENTAGE OF INTACT POLLEN

The percentage of intact pollen differs between species(KW = 22.2; d.f. = 5; P-value � 0.0005) (Fig. 4A).Removing R. communis from the dataset eliminatesthese differences (KW = 5,67; d.f. = 4; P-value < 0.25).The mean over all the media of the percentage ofintact pollen is > 60% for C. papaya, N. sylvestris and

Figure 2. Proportion of intact pollen as a function of the level of hydration. In all the plots, circles represent means,

error bars represent 95% confidence intervals and ‘N’ is the number of observations. The data are shown for all the spe-

cies pooled together (A) and for each species separately (B). The statistics for the result of the Kendall test are shown.

© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 478–490

POLLEN WALL MORPHOLOGY AND FUNCTION 483

J. integerrima. For M. tricuspidata and I. german-ica, the mean is between 40% and 60%. Ricinus com-munis has an average of < 40% of intact pollen, thelowest value of all the species investigated. The dataof intact pollen as a function of hydration level foreach species separately (Fig. 2B) show that R. com-munis has a relatively poor performance (< 50% ofintact grains) at all the levels of hydration except forthe lowest (1 M mannitol); M. tricuspidata andI. germanica are < 50% for the high levels of hydra-tion (0 and 0.2 M mannitol), but > 50% for the lowones; and C. papaya, N. sylvestris and J. integerrimaare somewhat > 50% of intact pollen for all themedia.

PERCENTAGE OF POLLEN WITH DISRUPTED PLASMA

MEMBRANE

We found significant differences between species inthe percentage of pollen with disruption of the cellmembrane (KW = 30.35; d.f. = 5; P-value < 0.001)(Fig. 4A). These differences hold if R. communis orJ. integerrima are removed from the dataset(KW = 13.3; d.f. = 4; P-value < 0.01 for R. commu-nis, and KW = 18.5, d.f. = 4, P � 0.001 for J. inte-gerrima), but not if both R. communis andJ. integerrima are removed (KW = 2.83; d.f. = 3;P-value < 0.5); therefore, these two species accountfor the differences observed. The data for each level

A B C D

E F G H

I J K L

Figure 3. Effects observed on the exine and on the plasma membrane, produced by the swelling of the grain. Pollen is

stained with Alexander’s stain, which colours the exine green and cytoplasm pink. A, B, Ricinus communis. A, Intact

hydrated pollen. B, Pollen with unimpaired exine and disrupted plasma membrane. The cytoplasm moves through the

aperture. C, D, Carica papaya. C, Intact pollen. D, Pollen with disruption of the plasma membrane. As in B, the cyto-

plasm moves through apertures. E, F, Iris germanica. E, Hydrated pollen. F, Pollen with disruption of the cytoplasmic.

G, Pollen of Nicotiana sylvestris with disrupted plasma membrane. H, I, Matthiola tricuspidata. H, Intact pollen. I, Pol-

len with disrupted plasma membrane. The cytoplasm moves through a tiny breakage of the exine. J–L, J. integerrima.

J, Intact pollen. K, Pollen with exine breakage, but unimpaired plasma membrane. L, Pollen with breakage of the exine

and disruption of the plasma membrane.

© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 478–490

484 A. MATAMORO-VIDAL ET AL.

of hydration separately (Fig. 4B) suggest that therelative differences between species in the percent-age of pollen with disrupted plasma membrane are

qualitatively similar for the high levels of hydration,as compared with the full dataset (Fig. 4A). It couldbe that these interspecific differences decrease for

%Pollen intact

Pollen with disruption of the cell membrane

Pollen with broken exine and unimpaired cell membrane

n = 4n = 12n = 16 n = 24 n = 12

CaricaNicotianaIrisRicinus Matthiola Jatropha

n = 4

0

20

40

60

80

100 or

Broken exine and unimpaired plasma membrane

n = 4 n = 4 n = 4 n = 4 n = 6 n = 6 n = 6 n = 6 n = 3 n = 3 n = 3 n = 3 n = 1 n = 1 n = 1 n = 1 n = 3 n = 3 n = 3 n = 3 n = 1 n = 1 n = 1 n = 1

0

10

20

30

40

%

Mannitol (M)0 0.2 0.45 1 0 0.2 0.45 1 0 0.2 0.45 1 0 0.2 0.45 1 0 0.2 0.45 1 0 0.2 0.45 1

CaricaNicotianaIrisRicinus Matthiola Jatropha

All media

Car

ica

Nic

otia

naIris

Ric

inus

Mat

thio

la

Jatro

pha

n = 4 n = 6 n = 3 n = 1 n = 3n

n = 1 n = 4 n = 6 n = 3 n = 1 n = 3 n = 1 n = 4 n = 6 n = 3 n = 1 n = 3 n = 1 n = 4 n = 6 n = 3 n = 1 n = 3 n = 1

0

Car

ica

Nic

otia

naIris

Ric

inus

Mat

thio

la

Jatro

pha

Car

ica

Nic

otia

naIris

Ric

inus

Mat

thio

la

Jatro

pha

Car

ica

Nic

otia

naIris

Ric

inus

Mat

thio

la

Jatro

pha

Disruption of the cell membrane or

Mannitol (M)0.2 0.45 1

0

20

40

60

80

100

%

A

B

C

Figure 4. Proportion of intact pollen (white circles), of pollen with disruption of the plasma membrane (black circles),

and of pollen with breakage in the exine only (grey circles) as a function of the species. For each species, the data are

shown for all the levels of hydration pooled together (A) and for each level of hydration separately (B, C).

© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 478–490

POLLEN WALL MORPHOLOGY AND FUNCTION 485

low levels of hydration (0.45 and 1.00 M mannitol),but this could not be tested rigorously because of lowsample sizes.

PERCENTAGE OF POLLEN WITH BROKEN EXINE AND

UNIMPAIRED PLASMA MEMBRANE

There were also interspecific differences in the per-centage of pollen with broken exine and unimpairedplasma membrane (KW = 53.6; d.f. = 5; P-value < 0.001) (Fig. 4A), due to the fact that thiseffect was observed only in J. integerrima andM. tricuspidata. These two species have neverthelessdifferent behaviours (Fig. 4C): J. integerrima has onaverage much higher percentage than M. tricuspi-data (J. integerrima: mean = 15.42; SD = 7.6; M. tri-cuspidata: mean = 2.7; SD = 3.44). In addition,M. tricuspidata has a roughly stable percentage forall the levels of hydration, whereas it is variable forJ. integerrima (Fig. 4C).

RELATIONSHIPS BETWEEN POLLEN MORPHOLOGY AND THE

EFFECTS INDUCED BY THE HYDRATION

The diminution of intact pollen grains was due tobreakage of the plasma membrane for all the speciesand to breakage of the exine in the inaperturate spe-cies (J. integerrima and M. tricuspidata) only(Fig. 4). Leaving aside exine breakage, which isobserved only in species lacking apertures, we testedfor an effect of the morphological properties on thepercentage of disruption of the plasma membrane.

We found significant differences in the percentageof pollen with disruption of the plasma membranedue to aperture pattern (KW = 18.15; d.f. = 3; P-value < 0.001), but these differences were completelyattributable to the omniaperturate pattern (J. inte-gerrima) (KW after removal of J. integerrima fromthe dataset = 3.97; d.f. = 2; P-value < 0.15).

We found differences between the exine ornamenta-tions patterns in their percentage of pollen with theplasma membrane disrupted (KW = 18.1; d.f. = 2, P-value < 0.001) (Fig. 5A). The percentage is highestfor the perforate ornamentation (C. papaya,N. sylvestris and R. communis), intermediate for thereticulate pattern (I. germanica and M. tricuspidata)and it attains its lowest value with the crotonoid pat-tern (J. integerrima). Testing differences betweenpairs of ornamentation patterns systematicallyresulted in significant differences: reticulate/perforate(KW = 3.9; d.f. = 1; P-value < 0.05); perforate/croto-noid (KW = 14.5; d.f. = 1; P-value < 0.001); reticulate/crotonoid (KW = 11.3; d.f. = 1; P-value < 0.001). Byconsidering each level of hydration separately wefound that, for all the levels of hydration, the croto-noid pattern has still the lowest percentage of pollen

with the plasma membrane disrupted relatively tothe perforate and reticulate patterns (data notshown).

There was a significant positive correlationbetween exine thickness and the percentage of pollenwith disruption of the plasma membrane (Kendalltest; s = 0.278; P-value < 0.002) (Fig. 5B). This ten-dency remained when looking at each level of hydra-tion separately (data not shown) and it could be thatthe effect of exine thickness is bigger for the highlevels of hydration (0 and 0.2 M mannitol) but thiscould not be tested because of the resulting lowsample sizes.

DISCUSSION

Understanding the relationship between structureand function of morphology is a longstanding ques-tion in biology (e.g. Lauder, 1981). In the case of pol-len, the involvement of wall morphology in theaccommodation of volume change was recognizedlong ago (Wodehouse, 1935). As the gametophyticphase represents a crucial stage in the life cycle ofplants, it is of interest to know how its fitness isaffected by environmental fluctuations. This studyfalls into such an approach, by allowing the mea-surement of how the structure of the pollen wallmay or may not accommodate changes in the volumeof the grain further to hydration and avoid pollenbreakage.

Samples that were exposed to high levels of hydra-tion had lower percentages of intact grains thansamples that were exposed to lower levels (Fig. 2).This indicates that our experimental conditions effec-tively induce a stress to the pollen in a dose-depen-dent manner: the higher the level of hydration thelower the percent of intact grains. The effectsobserved are thus due to the hydration induced byour experimental conditions and not by an experi-mental artefact. This stress might be similar to thoseencountered by pollen grains in natural conditions,for example, when they are exposed to rainfall orcovered in dew. Pollen viability is affected by suchenvironmental fluctuations (Lisci et al., 1994) and itmay be that pollen has been adapted to these varia-tions. One way to achieve this is to evolve a morphol-ogy that allows accommodating the increase ofvolume induced by abrupt hydration and swelling.Under this scenario, differences in the response tothe hydration are expected between pollen grainsexhibiting morphological differences.

The exposure to hydration caused inter-specific dif-ferences in the rates of wall breakage and/or of dis-ruption of the cell membrane breakage (Figs 3, 4).Exine breakage was seen only in pollen grains

© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 478–490

486 A. MATAMORO-VIDAL ET AL.

lacking apertures (M. tricuspidata and J. inte-gerrima), never in aperturate pollen. This suggeststhat apertures contribute to the accommodation ofvolume change due to abrupt hydration by avoidingbreakage of the exine. The percentage of pollen withexine breakage and unimpaired cell membrane wasmuch higher in J. integerrima than in M. tricuspi-data, suggesting that the wall of J. integerrima pol-len is much more delicate than the wall ofM. tricuspidata. Accordingly, we found that theexine of J. integerrima is nearly five times thinnerthan that of M. tricuspidata. We may conclude thatthe thin wall of J. integerrima does not tolerate anincrease of the volume of the cytoplasm.

The rate of breakage of the plasma membrane ofJ. integerrima was the lowest of all the species. Thepollen of J. integerrima is omniaperturate. This typeof pollen has an exine that is uniformly thin and anintine that is uniformly thick (Thanikaimoni et al.,1984; Furness, 2007). Thickness of the intine wall ofJ. integerrima could help to maintain integrity of theplasma membrane further to hydration.

The interspecific differences observed for the rateof breakage of the plasma membrane might berelated to exine ornamentation and thickness. Thereis a positive correlation between the rate of cellmembrane disruption and the thickness of the exine.Thin exine walls are more flexible (Katifori et al.,2010; and references therein) and thus could exertfewer constraints on the plasma membrane duringhydration. Regarding ornamentation patterns, thehighest rate of breakage of the plasma membranewas found for the perforate pattern, followed by thereticulate pattern and the crotonoid pattern. Theouter exine of the crotonoid pattern is made of unitsof exine (pilea) that are not closely connected witheach other. This facilitates a deformation that makesthe surface of the grain convex, as it is the casewhen pollen swells (Katifori et al., 2010). Conversely,the perforate pattern is composed of a network ofridges of exine with tiny openings. A high stiffness isthus expected for this morphology. The reticulatepattern may be expected to have an intermediateflexibility, as it is made of ridges of exine that are

Perforate (Carica + Nicotiana + Ricinus)

Reticulate (Iris + Matthiola)

Crotonoid (Jatropha)

1030

5070

Exine ornamentation

Dis

rupt

ion

of c

ell

mem

bran

e (%

)n = 12

n = 36

n = 24

2040

60

020

4060

80

n = 12

n = 4n = 4

n = 12

n = 16

n = 24

Dis

rupt

ion

of c

ell

mem

bran

e (%

)

Exine thickness (µm)

0.54 (Jatropha)

1.56 (Carica)

1.62(Nicothiana)

1.69 (Iris)

1.71(Ricinus)

2.38(Matthiola)

A

B

Figure 5. Proportion of pollen grains with disruption of the plasma membrane, as a function of exine ornamentation

pattern (A) and exine thickness (B). Circles are means made across all the media. Similar results were obtained by con-

sidering each level of hydration separately (not shown).

© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 478–490

POLLEN WALL MORPHOLOGY AND FUNCTION 487

firmly connected, but with relatively large openings.It could be that such differences in exine flexibilityplay a role in the avoidance of pollen breakage.

In this study, we have shown that there are quali-tative and quantitative differences between speciesin how their pollen responds to hydration. In a quali-tative point of view, it seems clear that the inapertu-rate, thin and crotonoid-like exine of J. integerrimapollen differs from the others species in way thatmake it much more delicate. However, it remains tobe established whether the quantitative differenceobserved among all species are attributable to differ-ences in pollen morphology. This could not be estab-lished unambiguously in this study because thesampled species differ from each other by too manyvariables (aperture pattern, exine ornamentationand thickness). Moreover, pollen water content playsalso a crucial role in pollen response to hydration.The water content of the pollen after anther anthesisis variable between species. In some species, pollenis released with a content of water greater than 30%[pollen partially hydrated, according to the terminol-ogy of Franchi et al. (2002)], whereas in other speciespollen has < 30% of water (pollen partially dehy-drated). The response of the grains to hydration islikely to depend on the initial water content: par-tially hydrated pollen having higher initial watercontent than partially dehydrated pollen, the lattermight be subject to higher stress than the former.Thus, the possibility that the effects observed resultboth from the morphological properties of the pollenwall and from the initial water content cannot beruled out. Further studies controlling for initialwater content and pollen morphology are required todisentangle the relative contribution of these two fac-tors to the capacity of survival of the grain further tohydration. We propose a model predicting pollenresponse to hydration depending on pollen morphol-ogy and water content. The validity of this modelremains to be tested, but it provides a roadmap forfuture experiments aiming to answer the question ofthe contribution of pollen morphological and physio-logical features to the accommodation of a volumeincrease and to the avoidance of pollen breakagefurther to hydration.

The model (Fig. 6) states that increased hygro-metry in pollen environment during the pollen dis-persal phase may have different effects on the grain,depending on water exchanges (driven by pollenwater content and hydrodynamics between the cyto-plasm and the environment) and on the rigidity andthe flexibility of the wall (driven by wall morphologi-cal properties). When pollen is placed in hypotonicconditions, cytoplasm eventually hydrates and itsvolume increases (Fig. 6A). Such hydration andvolume increase would depend on the initial water

content (Firon et al., 2012) and on the waterexchanges between the cytoplasm and the environ-ment, which are in part regulated by the apertures(Heslop-Harrison, 1979a). If there is water influx,then a swelling of the grain is expected. In this case,if the wall is plastic (i.e. capable of deformationthrough stretching), it will accommodate the swellingof the cytoplasm (Fig. 6B). Such plasticity is expectedin species with apertures, loose exine ornamentationpattern and thin exine (Katifori et al., 2010). If thewall is not plastic, the swelling of the cytoplasmmight be constrained by the wall (Fig. 6C). In thiscase, two configurations should occur, depending onthe strength of the exine and the intine wall. If theexine is delicate, the exine will break because of thestress induced by the swelling of the cytoplasm(Fig. 6D). This effect should be amplified if the intinewall is thick and robust. Such exine rupture due to asudden and large expansion of the grain has beenreported in Cupressus L. and in MontrichardiaCrueg., pollen of which has a thin exine and a thickintine (Weber & Halbritter, 2006; Chichiricc�o et al.,2009; Danti et al., 2011) and is also observed here inpollen of J. integerrima which presents similar char-acteristics. Alternatively, if the exine is rigid, it willconstrain the membrane of the cytoplasm and theintine (Blackmore & Barnes, 1986) and this will pro-voke the disruption of the plasma membrane(Fig. 6E, E0) if the difference in osmotic pressure onthe different sides of the membrane is high enough.This phenomenon, comparable to blowing a balloonconfined in a rigid box, should be amplified if theintine is thin and delicate. In the case of aperturatepollen, the disruption of the plasma membraneshould occur through an aperture (Fig. 6E), whereasin the case of inaperturate pollen, it should occurthrough local breakage of the exine (Fig. 6E0).

CONCLUSIONS

We present a method that allows to induce a stress topollen in a dose-dependent manner. This stress, ahydration resulting in a swelling of the grain, is simi-lar to those encountered by pollen grains in naturalconditions (Lisci et al., 1994; Pacini, 2000). Using thismethod to realize experimental observations on agroup of species differing in their pollen morphologyallowed us to demonstrate different responses, whichdepend to some extend on the morphology of the wall.In particular, the presence of apertures and the thick-ness of the wall seem to facilitate pollen volumeincrease and avoidance of wall breakage. From anevolutionary perspective, pollen wall morphologymight have played a role in the evolutionary successof eudicots (Furness & Rudall, 2004; Matamoro-Vidal

© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 478–490

488 A. MATAMORO-VIDAL ET AL.

et al., 2012). At least two selective pressures areknown to act on pollen morphology: one related to theefficiency of pollen germination and survival (Dajoz,Till-Bottraud & Gouyon, 1991, 1993; Till-Bottraudet al., 1999) and another one related to the retractionof the wall during dehydration (Halbritter & Hesse,2004; Volkova et al., 2013). We propose the existenceof an additional selective pressure that might be act-ing on pollen wall morphology through its influenceon survival upon hydration and swelling of the grain.

ACKNOWLEDGEMENTS

We thank T. Deroin, J. Dupont, T. Giraud, M. Lopez-Villavicencio, A. Ressayre and G. Restoux for helpfuldiscussions and advice. J. Doyle, C. Furness and J.Moustakas-Verho and an anonymous reviewer made

useful comments and corrections on an earlier versionof the manuscript. We thank E. Couradeau; C. Dje-diat; A. Dubois, C. Rausch, C. Sanchez, L. Saunoisand the ‘Plateforme de Microscopie et d’Imagerie duMNHN’ for valuable technical assistance. We thankthe Royal Botanic Gardens, Kew, and the Parc Botani-que de Launay, Orsay, for providing seeds and plantmaterial. This work was supported by the ‘ActionTransversale du Mus�eum Formes Possibles, FormesR�ealis�ees’ (Mus�eum national d’Histoire naturelle).

REFERENCES

Bergamini-Mulcahy G, Mulcahy DL. 1983. A comparison

of pollen tube growth in bi- and trinucleate pollen. In:

Mulcahy DL, Ottaviano E, eds. Pollen: biology and implica-

tion for plant breeding. New York: Elsevier, 29–33.

HydrationLow High

Cytoplasm

Exine

Aperture

Cell membrane disrupted

Exine disrupted

Plastic exine

Non-plastic exine

Rigid exine & delicate intine

Delicate exine & robust intine

A B

C

D

E

Intact pollen

cell membrane+ intine

E’

Cell membrane disrupted and local breakage of the exine

Favoured by:Low pollen initial water content Permeability of the pollen wall

Figure 6. Model predicting possible responses of pollen grains following hydration. A, During hydration, water eventu-

ally enter into the pollen grain and causes a swelling. B, If exine is plastic enough, the cytoplasm and the exine will

grow and reach an equilibrium state. All the structures remain intact. C, If the exine lacks flexibility, it constrains the

swelling of the plasma membrane and intine. D, If the level of hydration is high enough, the exine delicate and the

intine resistant enough, then the exine will break. E, E0. In the case that the exine is resistant and the intine is delicate,

then the plasma membrane breaks through an aperture (E) or through a local breakage of the exine (E0).

© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 478–490

POLLEN WALL MORPHOLOGY AND FUNCTION 489

Blackmore S, Barnes SH. 1986. Harmomegathic mecha-

nisms in pollen grains. In: Blackmore S, Ferguson IK, eds.

Pollen and spores: form and function. Linnean Society Sym-

posium Series. London and New York: Academic Press,

137–148.

Chichiricc�o G. 2007. Post-shedding dehydration and in vivo

temporal germination of Crocus pollen. Grana 44: 142–147.

Chichiricc�o G, Span�o L, Torraca G, Tartarini A. 2009.

Hydration, sporoderm breaking and germination of Cupres-

sus arizonica pollen. Plant Biology 11: 359–368.

Dajoz I, Till-Bottraud I, Gouyon P-H. 1991. Evolution of

pollen morphology. Science 253: 66–68.

Dajoz I, Till-Bottraud I, Gouyon P-H. 1993. Pollen aper-

ture polymorphism and gametophyte performance in Viola

diversifolia. Evolution 47: 1080–1093.

Danti R, Della Rocca G, Calamassi R, Mori B, Lippi

MM. 2011. Insights into a hydration regulating system in

Cupressus pollen grains. Annals of Botany 108: 299–306.

Edlund AF, Swanson R, Preuss D. 2004. Pollen and

stigma structure and function: the role of diversity in polli-

nation. Plant Cell 16: S84–S97.

Erdtman G. 1969. Handbook of palynology. New York: Haf-

ner Publishing Co.

Firon N, Nepi M, Pacini E. 2012. Water status and associ-

ated processes mark critical stages in pollen development

and functioning. Annals of Botany 109: 1201–1213.

Franchi GG, Nepi M, Dafni A, Pacini E. 2002. Partially

hydrated pollen: taxonomic distribution, ecological and evo-

lutionary significance. Plant Systematics and Evolution

234: 211–227.

Franchi GG, Piotto B, Nepi M, Baskin CC, Baskin JM,

Pacini E. 2011. Pollen and seed desiccation tolerance in

relation to degree of developmental arrest, dispersal, and

survival. Journal of Experimental Botany 62: 5267–5281.

Furness CA. 2007. Why does some pollen lack apertures? A

review of inaperturate pollen in eudicots. Botanical Journal

of the Linnean Society 155: 29–48.

Furness CA, Rudall PJ. 2004. Pollen aperture evolution –

a crucial factor for eudicot success? Trends in Plant Science

9: 154–158.

Halbritter H, Hesse M. 2004. Principal modes of infoldings

in tricolp(or)ate angiosperm pollen. Grana 43: 1–14.

Heslop-Harrison J. 1979a. An interpretation of the hydrody-

namics of pollen. American Journal of Botany 66: 737–743.

Heslop-Harrison J. 1979b. Pollen walls as adaptive systems.

Annals of the Missouri Botanical Garden 66: 813–829.

Heslop-Harrison Y, Heslop-Harrison J. 1982. The

microfibrillar component of the pollen intine: some struc-

tural features. Annals of Botany 50: 831–842.

Hesse M. 1999. Pollen wall stratification and pollination. In:

Dafni A, Hesse M, Pacini E, eds. Pollen and pollination.

Vienna: Springer, 1–11.

Katifori E, Alben S, Cerda E, Nelson DR, Dumais J.

2010. Foldable structures and the natural design of pollen

grains. Proceedings of the National Academy of Sciences of

the United States of America 107: 7635–7639.

Lauder GV. 1981. Form and function: structural analysis in

evolutionary morphology. Paleobiology 7: 430–442.

Lisci M, Tanda C, Pacini E. 1994. Pollination ecophysiol-

ogy of Mercurialis annua L. (Euphorbiaceae), an anemophi-

lous species flowering all year round. Annals of Botany 74:

125–135.

Matamoro-Vidal A, Furness CA, Gouyon P-H, Wurdack

KJ, Albert B. 2012. Evolutionary stasis in Euphorbiaceae

pollen: selection and constraints. Journal of Evolutionary

Biology 25: 1077–1096.

Muller J. 1979. Form and function in angiosperm pollen.

Annals of the Missouri Botanical Garden 66: 593–632.

Pacini E. 2000. From anther and pollen ripening to pol-

len presentation. Plant Systematics and Evolution 222:

19–43.

Payne WW. 1972. Observations of harmomegathy in pollen

of Anthophyta. Grana 12: 93–98.

Punt W, Hoen PP, Blackmore S, Nilsson S, Le Thomas

A. 2006. Glossary of pollen and spore terminology. Review

of Palaeobotany and Palynology 143: 1–81.

Scotland RW, Barnes HB, Blackmore S. 1990. Harmo-

megathy in the Acanthaceae. Grana 29: 37–45.

R Development Core Team. 2010. R: a language and envi-

ronment for statistical computing. Vienna: R Foundation for

Statistical Computing.

Thanikaimoni G. 1986. Pollen apertures: form and func-

tion. In: Blackmore S, Fergusson IK, eds. Pollen and spores:

form and function. London: Linnean Society, 120–134.

Thanikaimoni G, Caratini C, Nilsson S, Grafstr€om E.

1984. Omniaperturate Euphorbiaceae pollen with striate

spines. Bulletin du Jardin Botanique National de Belgique

54: 105–125.

Till-Bottraud I, Vincent M, Dajoz I, Mignot A. 1999. Pol-

len aperture heteromorphism. Variation in pollen-type pro-

portions along altitudinal transects in Viola calcarata.

Comptes Rendus de l’Acad�emie des Sciences – Series III –

Sciences de la Vie 322: 579–589.

Volkova OA, Severova EE, Polevova SV. 2013. Structural

basis of harmomegathy: evidence from Boraginaceae pollen.

Plant Systematics and Evolution 299: 1769–1779.

Weber M, Halbritter H. 2006. Exploding pollen in Mon-

trichardia arborescens (Araceae). Plant Systematics and

Evolution 263: 51–57.

Wodehouse R. 1935. Pollen grains, their structure, identifi-

cation and significance in science and medicine. New York:

McGraw-Hill.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article:

Table S1. Raw data for the analyses presented in this study.

© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 478–490

490 A. MATAMORO-VIDAL ET AL.