cupressus arizonica pollen wall zonation and in vitro hydration

Cupressus arizonica pollen wall zonation and in vitro hydration

G. Chichiricco,1 E. Pacini2

1Department of Environmental Sciences, University of L’Aquila, L’Aquila, Italy2Department of Environmental Biology, Botany Section, University of Siena, Siena, Italy

Received 28 February 2007; Accepted 4 September 2007; Published online 4 December 2007

� Springer-Verlag 2007

Abstract. The structure of Cupressus arizonicapollen at different degrees of hydration was examined

by using cytochemical staining and light (LM) and

scanning electron (SEM) microscopy. Most pollen

grains are inaperturate and a minority are provided

with an operculate pore enveloped by a concave

annulus. Intine consists of: 1) a thin polysaccharidic

outer layer, 2) a large polysaccharidic middle layer

that is spongy and bordered by a mesh of large and

branched fibrils, and 3) an inner cellulosic thick layer

with callose concentrated on the inner side, which

forms a shell around the protoplast. The protoplast is

egg-shaped with PAS positive cytoplasm and prom-

inent nucleus. Exine splits during hydration and is cast

off according to three major steps: 1) the split opens

like a mouth and the underlying intine is expelled by

swelling like a balloon, 2) the protoplast enveloped by

the inner intine is sucked in the outgrowing side, and

3) the backside of the intine gets rid of the exine shell.

In water containing salts, exine is rapidly released and

the middle intine may expand up to break the outer

layer, with disgregation of the spongy material and

release of the intine shell including the protoplast. In

water lacking salts, the sporoderm hydration and

breaking are negatively influenced by the population

effect. Pollen when air dried after the exine release

become completely flat owing to disappearance of the

middle intine layer which may be restored by dipping

pollen in water. The results are discussed in relation to

the functional potentialities of the sporoderm.

Keywords: Cupressus arizonica pollen; structure;

in vitro hydration; intine; exine

Introduction

Pollen from both gymnosperms and angiosperms

consist of the sporoderm and the included proto-

plast. Exine and intine walls are the components

of the sporoderm (Erdtman 1960), however the

former may be lacking, i.e. in aquatic plants such

as Callitriche truncata (Osborn et al. 2001), so

common pollen wall is the intine. Exine that is

made up of sporopollenin ensures protection

during pollen dispersal, and after pollination, it

controls the male-female interactions together

with the intine. Intine is pecto-cellulosic and plays

key roles in the cell germination and extension in

the pollen tube. During shedding, angiosperm

pollen is invariably two or three celled, whereas a

variable number of cells, generally from one to

five, occurs in gymnosperms (Pacini et al. 1999,

Fernando et al. 2005). Another typical difference

Correspondence: G. Chichiricco, Department of Environmental Sciences, University of L’Aquila, Via Vetoil, 67100 L’Aquila, Italy

e-mail: [email protected]

Pl Syst Evol 270: 231–242 (2008)

DOI 10.1007/s00606-007-0610-6

Printed in The Netherlands

Plant Systematicsand Evolution

concerns the pollen physiology; gymnosperm

pollen grains generally hydrate in the pollination

drop held on the ovule micropyle and slowly

germinate after carried in the nucellus (Fernando

et al. 2005), whereas angiosperm pollen both

hydrate and germinate soon after landing on the

stigma surface. In conifers the pollen wall varies

widely both in structure and function; it may or

may not have apertures, sculptures, wings, and

orbicules, and the exine may be released during

pollen hydration (Pacini et al. 1999, Fernando

et al. 2005). The pollen of Cupressus is particular

with regard to its wide structural stratification

(Pacini et al. 1999), but few details are available

on this matter (Grilli et al. 2000); on the contrary,

the pollen is extensively studied for its allergenic

properties (see Suarez-Cervera et al. 2003, Canini

et al. 2004, Mothes et al. 2004, Charpin et al.

2005).

In the present study, we examined Cupressusarizonica pollen at different degrees of hydration

by using cytochemical staining and light (LM)

and scanning electron (SEM) microscopy, with

the aim of contributing to its structure that could

be also useful for studies on pollinosis.

Materials and methods

Pollen collecting and storage. Pollen of Cupressusarizonica Greene was collected in February 2005 and

2006, from a tree growing on the avenue of the

University of L’Aquila (Coppito) (Italy). Branches

with mature pollen sacs were removed and left in the

laboratory until the pollen release at conditions similar

to those outside the laboratory, that is, 35–39% relative

humidity (RH) and 10–14�C. Pollen grains that were

not used at once were maintained for 1–2 days at

ambient conditions and then stored at –10�C.

Light and fluorescence microscopy. Whole

pollen grains were stained with: 1) IKI plus sulfuric

acid for detecting cellulose (Johansen 1940), (2)

calcofluor for b-glucans (Hughes and McCully 1975),

3) aniline blue for callose (O’ Brien and McCully 1981),

and 4) DAPI for DNA (Coleman and Goff 1985). The

images were acquired with a Zeiss Axioplan 2

fluorescence microscope equipped with Leica DFC

350 FX digital camera.

Pollen structure. Pollen grains for sections were

fixed in 3% glutaraldehyde in cacodilate buffer, or in

acetic alcohol (1:3) to avoid rehydration during

fixation, or becoming acetolyzed (Erdtman 1960).

The samples were dehydrated in an ethanol series and

embedded in Technovit 7100 resin (Kulzer). Sections

were cut at 2–5 lm with a Reichert-Jung 2040

microtome and stained with: 1) periodic acid-

Schiff’s (PAS) reagent for insoluble polysaccharides

(O’Brien and McCully 1981), 2) Schiff’s reagent plus

calcofluor for pectocellulosic walls (Mori and Bellani

1996), and 3) 0.05% toluidine blue for general

observations. The sections of acetolyzed samples

were not stained.

Pollen hydration. Pollen was hydrated at ambient

temperature (20�C) by storing in: 1) water lacking salts

(milliQ water), 2) tap water (waterP) (see Table 1 for

the composition), 3) solutions of calcium and sodium

ions alone dissolved in milliQ water, either as chloride

or bicarbonate salts at the same concentration as in the

waterP. The basic concentrations of pollen grain

(number/ml) were evaluated by counting the number

of pollen grains in a known water volume with a

hemocytometer (Nageotte chamber), and the

concentrations to use for each experiment were

obtained by dilutions. At different time intervals

after dipping in water, the pollen grains were

observed with the light microscope to monitor the

different steps of the sporoderm release, and to

determine variations in the pollen diameter with an

ocular micrometer at a magnification X 400. Usually,

50 pollen grains were measured for each test, and each

experiment was performed in triplicate.

Dehydration and rehydration of the pollenwithout exine. Pollen grains were dipped in 1–2

drops of waterP on a slide, and after they released

exine were air dried for some hours. Afterwards, they

were again hydrated as aforementioned.

Table 1. Composition (mg/L) of tap water (WaterP)

Ca++ 74.40

Mg++ 11.90

Na++ 4.80

K++ 1.35

NH4+ 0.020

F– 0.06

SO4–– 5.67

NO3– 9.51

NO2– <0.05

Cl– 6.33

HCO3– 273.28

232 G. Chichiricco and E. Pacini: Cupressus arizonica pollen wall zonation and in vitro hydration

Scanning electron microscope (SEM). Pollen

grains either living or dead were treated for SEM as

previously described (Chichiricco 2006).

Results

Light microscope (LM). Shed pollen grains are

unicellular, and when observed in immersion oil

(Fig. 1) are roundish but with some infoldings,

and also its cell is irregularly roundish. Pollen,

when mounted in oil several hours after shedding

(Fig. 2), appear deformed with evident infoldings

and contracted cell.

Sections of shed pollen. The sections of

pollen grains stain homogeneously with either

toluidine blue or Schiff’s reagent but the pollen

components are not well defined (Fig. 9),

independently of whether the grains were fixed

immediately after shedding or several hours

later. In the sections of acetolyzed pollen, a

minority of pollen grains displays an exine gap

filled with a plug that protrudes from the exine

layer (Fig. 8).

Pollen hydration. Pollen, when dipped in

water, soon become roundish and its protoplast

becomes egg-shaped. Thereafter, the exine wall

breaks by a linear slit and is released according to

the following steps (Fig. 26): 1) the slit opens like a

mouth and the underlying intine is expelled by

swelling like a balloon, 2) the protoplast is sucked

in the expanded side, 3) the backside of the intine

comes out from the mouth and the exine shell is

cast off. Following exine release, the protoplast

becomes either centrally or eccentrically arranged,

and the intine continues to expand up to break, with

dispersal of the pollen components in the medium.

The course of sporoderm breakage and pollen

expansion vary according to both the rehydration

medium and the pollen concentration in the

medium (Figs. 27, 28). As reported in Fig. 27,

when using waterP, the exine is released within

5–10 min and the intine breaks 24–48 h after.

When using milliQ water (Fig. 27), the exine

breaks after 15–60 min if the pollen concentration

is under 140,000 grains/ml, whereas it breaks not

before 48 h if the pollen concentration is over

200,000 grains/ml. Moreover, at concentrations

under 10,000/ml, the intine is subject to break

within 6–10 days, whereas it does not at higher

pollen concentrations. Whatever the concentration

of pollen, its size increment is highest when using

waterP (Fig. 28).

When using solutions with calcium or sodium

alone dissolved in milliQ water at the same

concentration as in the waterP, the breaking of

exine occurs either after a few minutes, if the ions

were as bicarbonate salts, or after many hours, if

the ions were as chloride salts.

Sections of pollen after the exinerelease. The sections of pollen deprived of exine

show well defined images of the intine stratification

(Fig. 10) in the: thin outer layer, large middle layer,

and thick inner layer including the protoplast with a

prominent nucleus. These components, with the

exception of the protoplast, are homogeneously

stained with either toluidine blue or Schiff’s reagent

(PAS), and the middle layer alternatively shows

granules (Fig. 10) with both stainings. The inner

intine layer is bright fluorescent after double

staining with Schiff’s reagent and Calcofluor

(Fig. 11).

Staining of whole pollen. Whole pollen

grains soon after staining with calcofluor, display

a hemispheric protuberance with yellowish

fluorescence (Fig. 3). Within 20–30 min from

staining, the exine generally breaks in the area of

the fluorescent protuberance, so this is pushed out

(insert of Fig. 4) by the swelling intine. Soon after

the exine release, intine layers emit a bright blue

fluorescence (Fig. 4), which is more intense in the

cytoplasmic side of the innermost layer; a bright

fluorescence diffuses in the medium if the outer

intine layer is broken by a slight squashing. The

protuberance induced by calcofluor is also evident

when observing with a light microscope, and it is

formed only after treatment with calcofluor.

After treating with aniline blue (Fig. 5), a

bright yellowish fluorescence is observed in the

inner intine layer, and more intensely in the inner

ring (not shown because not evident in the

photographs).

The double IKI-H2SO4 treatment stains blue

both the outer and the inner intine layers, the

latter much more intensely (Fig. 6), whereas the

middle layer does not stain as detected by

G. Chichiricco and E. Pacini: Cupressus arizonica pollen wall zonation and in vitro hydration 233

breaking the outer layer with a slight squashing;

no pollen component stains in the controls treated

with IKI alone (Fig. 7).

Pollen walls by SEM. Most pollen grains

(87,5%) of tested ones (80 pollen grains) showed

no aperture on the exposed surface of the exine,

and a minority (12,5%) exhibited a pore with

concave annulus and globose operculoid just

protruding from the pore (Fig. 12). Pollen,

following hydration, split along a precise linear

tract of exine, which is half of the pollen

circumference (Fig. 21). The splitting divides the

exine shell into two valves that open like a mollusc

bivalve to eject intine (Fig. 13), showing a

homogenous profile of the breaking surface (Fig.

21), without indentations or thinnings. Sometimes,

the exine and intine split together along the same

tract displaying their loose connection and their

different thicknesses (Fig. 21). Intine has a

compact and homogeneously smooth surface

(Fig. 14). This wall, when ruptured by a

prolonged hydration (Figs. 15, 17, 21), exhibits

its small thickness and the underlying middle layer

consisting of large branched fibrils (Fig. 16)

enveloping a spongy mass (Fig. 17). The spongy

layer disgregates and is progressively released in

the medium (Fig. 18) until becoming a net that

entangles the pollen shells (Fig. 20). The

disgregation exposes the inner intine (Fig. 19),

which appears as a massive egg-shaped shell with

rugose surface (Fig. 19). This wall retains the

integrity in water.

The pollen treated with calcofluor exhibit a

mass of fibrous material emerging from exine and

including orbicules (Fig. 23). This mass may

detach from exine and disperse on the pollen

surface (Fig. 22).

Harmomegathic changes. Shed pollen

grains when air dried decrease in volume by

the sporoderm inflexion, without significantly

reducing the diameter. When air drying after

the exine release, the middle intine completely

collapses and the outer intine flats without

contracting, until spreading as a veil over the

inner intine and around this to form an exact

disk (Fig. 24). The flat pollen grains dipped in

water swell and restore the roundish outline

(Fig. 25).

Discussion

Pollen morphology and structure. The pollen

of the Cupressaceae is characterized by a pore with

convex annulus, which plays key roles in the

pollen hydration and exine breaking (Van Campo

1953, Duhoux 1982, Bortenschlager 1990,

Kurmann 1994). The pore of living pollen is

crossed by a prominent operculoid that during the

pollen rehydration is pushed out by a transient

bulge of the intine (Duhoux 1982).

In our specimens of C. arizonica, only a

minority either of living or dead pollen grains is

provided with a germinal aperture consisting

of an operculate pore with concave annulus.

Figs. 1–11. Whole pollen grains (Figs. 1–7) and pollen grain sections (Figs. 8–11) of Cupressus arizonica.

LMs. 1, 2 Pollen grains in immersion oil soon after shedding (Fig. 1) or several hours later (Fig. 2). Note the

extensive zonation between the protoplast and the exine in Fig. 1, and the deformation of the sporoderm and the

protoplast in Fig. 2. Bar = 10 lm. 3, 4 Calcofluor-induced fluorescence before (Fig. 3) and soon after (Fig. 4) the

exine release. Note in Fig. 3 the fluorescence of the cup-shaped protuberance (arrows), in Fig. 4, the fluorescence

of the intine, of the underlying layer and of the inner wall; in the insert, the fluorescence of the detached

protuberance. Bar = 50 lm. 5 Aniline blue induced fluorescence in the inner pollen wall (arrows) after the exine

release. Bar = 25 lm. 6, 7 Blue staining of the outer intine (OI) and the inner intine (arrow) after treatment with

IKI plus sulfuric acid (Fig. 6), and control (Fig. 7). Bar = 40 lm. 8 Section of acetolized pollen grains showing

exine aperture closed with the operculoid (arrows). E = exine. Bar = 20 lm. 9, 10 Pollen grains sectioned some

hours after shedding (Fig. 9) or after the exine release in water (Fig. 10), both stained with PAS reagent. Note the

indefinite pollen components in the first section, and the well defined outer intine (OI), middle intine (MI) and

inner intine (arrow) in the second section. Bar = 10 lm in Fig. 9 and 20 lm in Fig. 10. 11 Section of exine free

pollen stained with Schiff’s reagent and calcofluor; note the fluorescence of the inner intine. Bar = 20 lm

b


Figs. 12–17. SEM observations of pollen grains of Cupressus arizonica during in vitro hydration.

Bar = 10 lm. 12 Detail of the pollen surface showing numerous orbicules and a pore (arrow) with concave

arnulus and operculoid. 13 Breaking and opening of the exine (E) with ejection of the swelling intine. 14 Swelled

pollen after exine release. 15 Pollen ejection from the exine (E) with simultaneous splitting of the outer intine

(OI) and exhibition of the middle intine (MI). 16, 17 Middle intine (MI) showing in Fig. 16 the branched fibrils

and in Fig. 17 the spongy mass


Figs. 18–23. 18, 19 Disgregation of the middle intine with partial (Fig. 18) and complete (Fig. 19) release of the

inner intinous shell which encloses the protoplast. 20 Net around the exine shells, arising from disgregation of the

middle intine. 21 Exine (E) and outer intine (OI) simultaneously and correspondingly broken showing their

different thicknesses and their detachment after discharge of the midle intine. 22, 23 Pollen treated with

calcofluor. Note in Fig. 22 both the bulge emerging from exine and the bulge dispersion on the pollen surfaces

(arrows), and in Fig. 23 a particular of the bulge consisting of fibrous material. Bar = 5 lm in Fig. 22 and 2 lm

in Fig. 23


Likewise, a plugged exine gap is sometimes

evidenced in the sections of acelosized pollen. A

common feature after calcofluor treatment is the

formation of a fluorescent globose bulge, as

already reported by Duhoux (1982). However, in

the present case it is persistent, and when

observed by SEM it seems to be formed by a

material coming from the inside of the pollen,

which polymerizes and inglobates orbicules

during its release from the exine. Thus, it is

comparable to the pectic emission of the angio-

sperm germinal apertures (Heslop-Harrison and

Heslop-Harrison 1985). If that is the case,

however, two major points are questionable: 1)

the bulge forms only when pollen is rehydrated in

calcofluor, and 2) it is present in all pollen grains

despite only a minority of them is porate.

Particular attention deserves the intine.

According to the three-layered model (Heslop-

Harrison and Heslop-Harrison 1991), the intine

has a variety of structural and physiological

specializations and consists of an outer homoge-

neous layer and a middle inhomogeneous layer

(tubular, columnar or lamellar) both rich of

pectins, and of an inner layer rich of cellulose.

Our observations by SEM concerning the

morphology and the dynamics of release of the

intine layers, together with the staining tests, are

compatible with such a model.

The outer layer is about three time thinner

than the exine and is homogeneous and compact.

Figs. 24 and 25. Exine free pollen air dried. Note the outer intine (OI) spread around the inner intine shell to

form a disk surrounded by orbicules. Bar = 20 lm. 25 The same as in Fig. 24 after rehydration. Note the swollen

outer intine (OI) and the exine shell. Bar = 10 lm

Fig. 26. Stages of the pollen hydration. Note in

succession: the exine splitting, the intine swelling, the

displacement of the protoplast, and the exine release


The high plasticity of this layer allows pollen to

triplicate its diameter by hydration. It gives

positive reactions with stainings for polysaccha-

rides including that for cellulose (IKI), however,

the moderate mechanical resistance excludes a

cellulosic nature.

The middle layer is very thick and viscous,

with the polysaccharidic components strongly

stainable either with calcofluor or Schiff’s

reagent (PAS). It is spongy when observed by

SEM, and enveloped by a mesh of large and

branched fibrils resembling cellulose macrofi-

brils. Such organization confers high hydrody-

namic properties and scarce mechanical

resistance to the middle layer, which result in

exceptional harmomegathic changes. It is

remarkable as this layer, in absence of the exine,

either may become insubstantial in dry state to

make the dehydrated pollen flat like a disk, or

may restore the roundish outline after immersion

in water. These features reflect the strong muci-

laginous nature and the mechanical deficiency of

Fig. 27. Pollen rehydration in water containing salts (water P) (A) and in water lacking salts (milliQ) (B, C) by

using different pollen concentrations. When using water P (A) the sporoderm walls break with release of the

protoplast within 24–48 h, regardless of the pollen concentration. When using water milliQ, the sporoderm walls

are broken within a number of days if the pollen concentration is under 10,000 grains/ml (B), whereas the exine

only breaks if the pollen concentration is between 22,000–560,000 grains/ml (C)


the middle layer, which do not allow to hypoth-

esize a cellulosic framework.

The inner layer is the persistent wall of the

sporoderm. It is fluctuating within the mucilaginous

layer, and becomes well defined and thickened

during rehydration, parallel to the protoplast expan-

sion into an egg shape. Its polysaccharidic compo-

sition, based on the stainings tests, includes both

cellulose and callose with the latter concentrated on

the inner side. When observing by SEM, it is

compact and rugose resembling a nut shell. Such

intinous shell safeguards the protoplast, especially

during the harmomegathic changes which follow

the exine release, by replacing the mechanical

functions of the exine. At the same time, the

structural polarity predisposes pollen for the pollen

tube emission by the inner callosic layer.

The protoplast is weakly stainable, especially

in dry state and, as typical of the Cupressaceae

pollen (Fernando et al. 2005), it lacks insoluble

polysaccharides and includes a prominent nu-

cleus. Some traits such as the elongated shape

associated to the callosic wall, are signs of a

predisposed cell germination that do not meet

with the slow germination of conifer pollen, nor

with some factors ascribed to the slowness, such

as the absence of callose in the pollen tube wall

(see Fernando et al. 2005); in Pinus pinea pollen

(Pardi et al. 1996), the cytoplasm is rich of

callose for the pollen germination.

The morphological and cytochemical features

above described by LM and SEM agree, to some

extent, with studies by TEM on C. sempervirens(Grilli Caiola et al. 2000).

In vitro hydration. The absorbing capability

of the middle intine exceeds either the

mechanical resistance of the exine, or the

extensibility of the outer intine, so that pollen

may suck water up to break the sporoderm. The

exine, according to our observations by SEM,

breaks by a linear split that divides exine in two

valves with no indentations or thinnings along the

edges, which would indicate start points or

preferential pathways for the breaking. When

rehydration occurs in water containing ions such

as the waterP, the pollen concentration in the

water does not influence hydration and the exine

breaks after a few minutes, followed by intine

breaking in the next hours. When using milliQ

water, the hydration is negatively related to the

population effect. Both the temporal and the final

increments of pollen size are significatively

0

10

20

30

40

50

60

70

80

1 24 48

Time (h)

(rete

maidnello

Pµ

)m a b

a

b

a b

a

b a b

Fig. 28. Increasing diameter of pollen following hydration in water P (dotted) and in water milliQ (blank) by

using pollen concentrations of 200,000 grains/ml (a) and 5,000 grains/ml (b). The pollen diameter before

hydration was 26.4 lm


higher when hydrating in waterP than in milliQ

water, regardless of the pollen concentration.

Salts of both sodium and calcium have different

effects on the pollen rehydration according to

whether they are as chloride or bicarbonate.

These observations point out that the dynamics of

the pollen hydration is influenced by several

factors and the interactions of the mucilaginous

layer with the ionic species dissolved in water

play a key role.

Conclusion

The shedding pollen grains of Cupressus arizo-nica are unicellular microspores with highly

specialized sporoderm. This implies different

functionality as compared with other conifers,

as well as with angiosperm pollen. In particular,

the exine is a protective and weakly elastic wall

which, independently on whether it is or is not

provided with germinal aperture, is predisposed

for breaking during hydration. The intine with the

outer plastic layer enables pollen to expand and

to get rid of the exine. The middle mucilaginous

layer, despite being the largest pollen compart-

ment, lacks a substantial mechanical support and

represents the water sucker of the pollen, with

potential sugar reserves. The inner intine is the

real wall of the protoplast with structural and

physiological roles which support the cell viabil-

ity and germinability.

Such pollen wall zonation with shared and

coordinated roles makes the sporoderm highly

competent to control the hydrodynamics and the

pollen germination.

These observations stimulate further studies

to well understand the ultrastructure and compo-

sition of the sporoderm, which show some

questionable features that may be misinterpreted.

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cupressus arizonica pollen wall zonation and in vitro hydration

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