REVIEW

Pollen vacuoles and their significance

Ettore Pacini • Cedric Jacquard • Christophe Clement

Received: 5 April 2011 / Accepted: 8 June 2011 / Published online: 26 June 2011

� Springer-Verlag 2011

Abstract Vacuoles of several types can be observed in

pollen throughout its development. Their physiological

significance reflects the complexity of the biological pro-

cess leading to functional pollen grains. Vacuolisation

always occurs during pollen development but when ripe

pollen is shed the extensive translucent vacuoles present in

the vegetative parts in previous stages are absent. Vacuole

functions vary according to developmental stage but in ripe

pollen they are mainly storage sites for reserves. Vacuoles

cause pollen to increase in size by water accumulation and

therefore confer some degree of resistance to water stress.

Modalities of vacuolisation occur in pollen in the same

manner as in other tissues. In most cases, autophagic

vacuoles degrade organelles, as in the microspore after

meiosis, and can be regarded as cytoplasm clean-up fol-

lowing the transition from the diploid sporophytic to the

haploid gametophytic state. This also occurs in the gener-

ative cell but not in sperm cells. Finally, vacuoles have a

function when microspores are used for pollen embryo-

genesis in biotechnology being targets for stress induction

and afterwards contributing to cytoplasmic rearrangement

in competent microspores.

Keywords ER � Lysosomes � Pollen � Reserves �Vacuoles � Volume increase

Introduction

Vacuoles are absent in procaryotic cells but present in

algae, fungi and terrestrial plants (Margulis and Schwartz

1982). In algae and fungi they are always present except in

resting spores (Dottge 1973). Mosses show vacuoles in all

cell types, even in the dispersing spores (Neidhart 1979;

Brown and Lemmon 1981). In Pteridophyta, such as

Matteuccia and Equisetum, ripe spores contain vacuoles; in

others such as Polypodium vulgare vacuoles are absent and

water content is around 5% (Cran 1979). In other terrestrial

plants, vacuoles are reduced in meristems and practically

absent in ripe pollen and seeds, namely in reproductive

structures dispersed with a low water content in an arrested

developmental state (Heslop-Harrison 1979; Footitt and

Cohn 2001). Nevertheless vacuoles are present in seed and

pollen at various stages of development as well as during

their germination (Heslop-Harrison 1987; Hicks et al.

2004).

Vacuole functions vary according to cell differentiation

stage. The most common and widespread is regulation of

water potential in the cytoplasm under normal conditions

and stress (Maurel 1997; Kjellbom et al. 1999). Other

recognised functions are (i) storage of different types of

substances, such as carbohydrates and proteins or catabo-

lites (John 1991); (ii) turnover of cytoplasm parts by

lysosomes (Marty 1999); (iii) to drive plant cell growth

during differentiation (Maurel 1997). The mode of vacu-

olisation and the function of vacuoles in germinating pollen

were revealed by Hicks et al. (2004).

Despite the importance of vacuoles in plant cell physi-

ology, the process of vacuolisation during pollen devel-

opment has been described only incidentally by a few

authors (Cass and Karas 1975; Pacini and Juniper 1984;

Evans et al. 1992; Kuang and Musgrave 1996; Pacini et al.

E. Pacini

Dipartamento di Scienze Ambientali « Giacomino Sarfatti »,

Universita degli Studi di Siena, via PA Mattioli 4,

53100 Siena, Italy

C. Jacquard � C. Clement (&)

URVVC, UPRES EA 2069, Laboratoire de Stress, Defenses et

Reproduction des Plantes, Universite de Reims Champagne

Ardenne, BP 1039, 51687 Reims Cedex 2, France

e-mail: christophe.clement@univ-reims.fr

123

Planta (2011) 234:217–227

DOI 10.1007/s00425-011-1462-4

1992; McCormick 2004), while studying other aspects.

Special attention was recently paid to vacuoles throughout

pollen development and the results suggest that vacuoles

are plastic compartments and their aspects, origin and

functions vary during microspore/vegetative cell develop-

ment (Yamamoto et al. 2003).

The aim of this paper is to review current knowledge of

this process and to focus on the functions of vacuoles at

various steps of pollen ontogenesis in different groups.

Vacuole functions, activities and their manner of formation

are not only important for basic knowledge but also for

applied research, such as pollen embryogenesis (Touraev

et al. 1997), in vitro pollen development (Benito-Moreno

et al. 1988) and ripe pollen conservation (Yamamoto et al.

2003).

Vacuoles and pollen development

The process of vacuole formation in Angiosperm species

was followed from tetrad to ripe pollen stage (Fig. 1).

Except in orchids, a huge vacuole develops in the micro-

spore between tetrad release and the first pollen mitosis.

More precisely, the vacuolisation process concerns the

cytoplasm of meiocytes, microspores and vegetative cells

but not sperm cells. Some differences, for example the

number of waves of and peaks of vacuolisation are evident

in the species studied. Lycopersicum peruvianum and Olea

europaea have two peaks of vacuolisation, one in late

microspore stage when many small vacuoles forms, and the

other in middle bicellular stage when only one vacuole

forms (Fig. 1). In Prunus avium, Smilax aspera and Lilium,

the vacuolisation peak occurs at first haploid mitosis with

one big vacuole (Pacini and Franchi 1983; Pacini et al.

1986). Vice versa in Arabidopsis thaliana, Lolium perenne,

Zea mays and all grasses, it occurs in late microspore stage

with one big vacuole (Pacini and Keijzer 1989; Pacini et al.

1992).

In Lilium the vacuole develops from autophagic lyso-

somes after meiosis and occupies most of the microspore

volume just before mitosis (Clement et al. 1994). Soon

after mitosis, the vacuole is restricted to the vegetative cell

and reduces as starch accumulates. In the above species,

vacuoles, as extensive translucent areas, are not present in

ripe pollen though small vacuoles containing phytate can

be detected in some cases (Clement and Audran 1996).

Orchids are a totally different case for two reasons:

(i) vacuoles always destroy small portions of cytoplasm

and extensive vacuolisation is not observed; (ii) small

vacuoles with translucent content are present throughout

development and also in ripe pollen (Schlag and Hesse

1992; Pacini and Hesse 2002 and references therein).

Fig. 1 Scheme (not to scale) showing the extension of vacuolisation

(black areas) during pollen development of some representative

angiosperm species. Data of the authors and the literature cited

vacuolisation may occur in microspore, vegetative and generative

cells. A process of vacuolisation occurs in all species but peaks at

different stages of development. Vacuoles (extensive translucentareas) are absent from ripe pollen except that of orchids. Orchids are

a peculiar type because vacuoles are always small. Lycopersicumperuvianum and Olea europaea have two peaks of vacuolisation, one

during late microspore stage and the second during middle bicellular

stage. Prunus avium, Smilax aspera, Arabidopsis, Lilium, Loliumperenne, Zea mays and all the grasses only have one. Its apex is at first

haploid mitosis in Prunus and Smilax and in late microspore stage in

Lolium. GC generative cell, VC vegetative cell, SC sperm cell

218 Planta (2011) 234:217–227

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Pollen development inside the anther is generally quite

synchronous, but pollen mitosis and the peak of vacuoli-

sation are asynchronous. Large differences were observed

in an aneuploid cultivar of olive, leading cytoplasmic

degeneration in microspores and bicellular pollen (Pacini,

unpublished data). On the other hand, the synchrony of

vacuolisation has a maximum in orchids with pollinia,

probably because of the presence of cytomictic channels up

to early bicellular stage (Pacini and Hesse 2002).

Vacuole origins

Plant vacuoles are essentially produced in the following not

mutually exclusive ways (Fig. 2): by endoplasmic reticulum

(ER) and Golgi vesicles, by different activities of ER and by

lysosomes (Marty 1999). Vacuoles observed during pollen

development have different origins: (A) ER cisterns dilate to

form small vacuoles with clear contents which may fuse to

create larger vacuoles; (B) ER cisterns sequester portions of

cytoplasm and the content is subsequently destroyed;

(C) lysosomes, either simple or complex, combine cases

(A) and (B); (D) dictyosome vesicles fuse, increase in size,

or fuse with ER cisternae in case (B) and (C).

These modalities commonly destroy ribosomes, and

may destroy other organelles such as mitochondria and

plastids. This has been documented sporadically in

microspore cytoplasm, during the phase from sporophyte to

gametophyte. It was described in further detail in the

generative cytoplasm of Prunus avium (Pacini et al. 1986)

and Lolium perenne (Pacini et al. 1992). It has been

interpreted as elimination of plastids enabling maternal

inheritance, but parts of cytoplasm containing ribosomes or

other organelles may be destroyed. Vacuoles are always

small in orchids even if all the modalities previously

described above can be recognised.

When vacuoles originate according to the modalities

(B) and (C) the destroyed part is evident for a while inside

it and at different stages of destruction. Ripe vacuoles, i.e.

when the destruction of cytoplasm is over, sometimes have

granular contents and in some cases PAS (periodic acid

Schiff) and PATAg (periodic acid–silver proteinate) posi-

tive contents which later become translucent (Fig. 3).

The term ‘‘vacuolated stage’’ was used as a term for a

stage development when vacuolisation is extensive

(Christensen and Horner 1974; Blackmore and Barnes

1990), which must not be considered correct because the

bulk of the process may occur at different stages of

development; besides, at least in Olea europaea and

Lycopersicum peruvianum there are two vacuolisation

peaks (Fig. 1).

Pollen is internally and often externally polarised

because of the presence of apertures. It is not clear whether

vacuolisation in a given species always has the same pat-

tern or whether it varies from grain to grain. This question

arises from certain images of Lilium microspores showing

two extremely symmetrical vacuoles with respect to the

external geometry of the grain (Fig. 4). The peak of

a

b

c

d

Fig. 2 Vacuole biogenesis in plant cells. a ER cistern dilates at the

extremity to form small vacuoles (stars) with a clear content. b ER

vesicles sequestrate portions of cytoplasm and the content is

subsequently destroyed. C cytoplasm fraction following encapsulation

by lysosomes. c Secondary lysosomes, with internalised portion of

cytoplasm (stars) that is progressively digested. d Dictyosome

vesicles fuse between themselves increasing in size or fuse with ER

cisternae in the case b and c

Fig. 3 TEM section of a young microspore in Lilium showing

PATAg stained material (arrowheads) accumulated within the

developing vacuole. M microspore, L locular fluid, MW microspore

wall. Bar 20 lm

Planta (2011) 234:217–227 219

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vacuolisation with a single big vacuole is similar in all the

grains but it is not clear what happens in other stages when

there are many vacuoles. It is not known whether the

cytoskeleton always acts in the same way.

Vacuoles and transition from diploid to haploid state

The process of meiosis leads to the formation of

microspores with strong rearrangement of the cytoplasm

after meiosis due to the transition from diploid to the new

haploid state of the nucleus (Sangwan 1986; Sangwan et al.

1989). This phenomenon is initiated during meiosis and is

characterised by progressive hydrolysis of the cytosol and a

few organelles in lysosomes that grow and fuse to form the

vacuole (Yamamoto et al. 2003). It appears that autophagic

flow may discharge old cell components into the central

vacuole by a protrusion process, and thus contribute to the

‘‘cell turnover’’ that occurs during transition from the

sporophyte to gametophyte (Mckenzie et al. 1967). These

authors showed that during prolonged meiotic prophase,

ribosomes or cytoplasmic RNA are eliminated. This

observation is probably related to the sporophyte–game-

tophyte transition. The gene expression linked to the dip-

loid state is thus partly suppressed and the new haploid

genome controls gametophytic rather than sporophytic

development (reviewed in Borg et al. 2009). Little is

known about the enzymes involved in the turnover of

cytoplasmic RNA, though certain evidence suggests that

hydrolytic enzymes such as phosphatases may be involved

(Sangwan et al. 1989). In Datura innoxia, evidence for

digestion of cytoplasmic structures, such as ribosomes,

organelles and membranes during vacuolated microspore

stage and lytic activity within vacuoles were described. In

Lilium, lytic vacuoles can be easily seen in the cytoplasm

of microspores from meiosis until complete vacuolisation:

vacuoles develop from concentric ER cisternae (Fig. 5a)

and then contain various organelles (Fig. 5b, c) that are

progressively digested. In early bicellular pollen and during

maturation, lytic activity was mainly observed in small

vacuoles.

Vacuoles and reserve accumulation

After pollen mitosis, the fate of the large vacuole varies

according to species. In some cases, transitory vacuoles are

formed in the vegetative cell, split from the huge vacuole

that developed into the microspore (Pacini and Juniper

1984). In other plants, the huge vacuole of the microspore

completely disappears and small vesicles are produced de

novo by dictyosomes and rER and may be considered as

storage vacuoles (Yamamoto et al. 2003).

Fig. 4 TEM section of a young microspore in Lilium. Developing

vacuoles (V) are symmetric around the nucleus (N). Bar 8 lm

Fig. 5 TEM section of a tetrad spore in Lilium. a Concentric ER

saccules (arrowhead) and young lytic vacuoles (V) in the cytoplasm

at premeiosis. N nucleus, P plastid. Bar 10 lm. b Autophagic vacuole

(V) at meiosis formed from ER concentric saccules (arrowhead)

including mitochondria (m). Bar 30 lm. c Lytic vacuoles (V) sur-

round cytoplasm portion including organelles such as mitochondria

(m) or lipid globules (white star). C callose, P plastid. Bar 10 lm

220 Planta (2011) 234:217–227

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Some small translucent vesicles with electron opaque

cores are often observed in mature pollen (Fig. 6). Helsper

et al. (1984) and Butow et al. (1997) found phytate inside

them in Lilium and Chlorophyton pollen. They are involved

in providing inorganic reserves for germination but they do

not seem to give rise to vacuoles at germination (Baldi

et al. 1987). In Eryngium campestre, P particles including

polysaccharides have been described in the vegetative cell

during pollen maturation (Weber 1988). Using cryo-fixa-

tion/freeze substitution, Yamamoto et al. (2003) described

membrane-bound structures containing fibrillar substances

(MBFs) in mature pollen of Arabidopsis thaliana. These

structures are regarded as protein storage vacuoles for

further pollen germination. These special vacuoles origi-

nate from the activity of rough endoplasmic reticulum

(rER) that synthesizes large quantities of proteins that are

stored in the cytoplasm after formation of sperm cells. If

pollen is not dispersed after anther opening, these vacuoles

accumulate lytic enzymes and contribute to degradation of

the vegetative cell.

Various types of vacuoles/vesicles may be found in the

vegetative cell during pollen maturation. Hence they differ

in content and subsequently in function. For example, three

types of vesicles/vacuoles coexist in mature pollen of

Arabidopsis thaliana (Yamamoto et al. 2003): (i) P parti-

cles including polysaccharides that may be used for pollen

tube growth, (ii) lysosomes containing enzymes that take

part in pollen tube penetration of the female part and, (iii)

rER cisternae containing proteins that are also involved in

pollen tube elaboration.

At the onset of prophase, plastids are commonly undif-

ferentiated but accumulate starch and become amyloplasts

once or twice during development (Clement and Pacini

2001). In most species, there is only one phase of amylo-

genesis and this starts just after the peak of vacuolisation,

suggesting that sugars of vacuoles may be translocated to

plastids until needed for microspore/pollen metabolism.

Vacuoles are commonly absent in ripe pollen, and this is

very well documented when quantitative cytology is cou-

pled with three-dimensional reconstruction of organelles,

as in Plumbago zeylanica (Russell 1984) and Zea mays

(McConchie et al. 1987). Another clear demonstration of

the absence of vacuoles in ripe pollen is that of Evans et al.

(1991) who used pollen collected directly from the open

anther with a pollen collector. In many other cases, pollen

was fixed for observation while still in the anther where the

process of dehydration of pollen and anther is not com-

plete. This is probably why Theunis et al. (1991) found

vacuoles with translucent content in the vegetative cyto-

plasm of Spinacia oleracea pollen. Similarly, Chang and

Neuffer (1989) stated that in ripe Zea mays pollen, the

contents consists of a large centrally located vacuole and

the cytoplasm is pressed thinly between the vacuole and the

inner wall. These misinterpretations probably spring from

the fact that only shed pollen can be considered ripe; when

pollen is still in the anther it is difficult to determine

whether it is ripe or not. This means that real mature pollen

is only found when anthers are open and the pollen is in

equilibrium with the environment.

Vacuole and pollen volume increase

The growth of pollen during development is closely corre-

lated with vacuole development but occurs at different rates

in different plants (Fig. 1). Especially in some species, the

volume increase is greater in the final stages of development.

For example, in Cucurbita, the higher pollen volume the

grater the increase in vacuole volume during development

(Pacini 2000). In orchids, the increase in pollen volume and

development of vacuoles are limited (Pandolfi et al. 1993).

In most plant cells, the increase in vacuole volume is

correlated with water accumulation under the control of

aquaporins on plasma membranes and vacuoles (Maurel

1997; Kjellbom et al. 1999). This entry of water into vacu-

oles is linked to an increase in osmotic pressure in the vac-

uolar sap, due to accumulation of osmolites through the

tonoplast. In the case of pollen grains, the main growth of the

vacuole occurs when the microspores develop locular fluid

with large amounts of soluble and insoluble polysaccharides

arising from dissolution of callosic tetrad walls and from

tapetum secretions (Clement et al. 1998; Aouali et al. 2001).

It is therefore likely that the formation of the huge vacuole

leading to microspore and pollen size increase corresponds

to water and osmolite accumulation in vacuolar sap between

tetrad release and the first pollen mitosis.

Vacuolisation is followed by an increase in pollen grain

volume, but during ripening this volume decreases due to

Fig. 6 TEM section of vegetative cell in the mature pollen grain of

Lilium. Vacuoles (arrowheads) are small and include dense deposits.

L lipid, M mitochondrion, P plastid, PW pollen wall. Bar 20 lm

Planta (2011) 234:217–227 221

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water loss (Pacini 2000), raising the question of what fills the

space left by vacuolisation. Destroyed cytoplasm is probably

replaced by formation of new cytoplasm. No quantitative

data is available on cytosol increase during pollen devel-

opment, but we presume that this increase is largely due to

storage of materials such as lipids (spherosomes) and car-

bohydrates, present in plastids or the cytoplasm (Franchi

et al. 1996). Unlike in seeds, extensive protein storage does

not seem common during pollen ripening (Pacini 1996).

Vacuoles and pollen water content during maturation

Pollen develops inside the loculus, in a fluid that conveys

nutrients from the sporophyte to the developing gameto-

phyte. The flow of these substances decreases and ceases

after tapetum degeneration. The locular fluid thus disap-

pears before anther opening, allowing pollen exposure to

dispersing agents (Pacini and Hesse 2004). Pollen may lose

or gain some water during exposure depending on the

environment (Pacini 2000).

It was recently shown that it is possible to distinguish two

pollen types according to water content at shedding: pollen

partially dehydrated (PDP) has a water content less than

30%, partially hydrated pollen (PHP) has a water content

over 30% (Nepi et al. 2001; Franchi et al. 2002). PDP loses

much water before or as soon as it is exposed and resists

better to environmental stresses such as temperature and low

relative humidity better that PHP. However it takes longer to

rehydrate and germinate on the stigma. PHP loses little water

before exposure and is more vulnerable to environmental

stresses; on the other hand it germinates more quickly on the

stigma because little water needs to be absorbed (Pacini and

Hesse 2004). Specific genes are expressed during pollen

hydration (Wilson et al. 1997; McCormick et al. 1994),

indicating that pollen water balance is under genetic control.

Both classes of pollen are devoid of extensive vacuoles.

In wheat (PHP), cyclosis occurs in ripe pollen (Heslop-

Harrison et al. 1997). The absence of vacuoles means that

PHP stores surplus water in the cytoplasm as free molecules

or associated with certain polymers, such as carbohydrates

and proteins. Since water is the best known fluid stored in

vacuoles of plant cells (Maurel 1997; Kjellbom et al. 1999),

aquaporins are probably involved in accumulation of water

in the microspore vacuole during development under opti-

mal conditions and during drought stress.

Vacuoles, variations in osmotic potential and water

stress during pollen development

The osmotic pressure in vacuoles of microspore/pollen is

regulated during pollen development. It fluctuates between

350 and 450 mosm/l during microspore vacuolisation

(Hoekstra et al. 1993), showing that (i) the process of

vacuolisation is linked to maintenance of strong osmotic

pressure in vacuolar sap and that (ii) the microspore vac-

uole presumably adapts to external variations that would

modify pressure in the immediate environment.

Male gametophytes have low tolerance to environmental

variations. For example, plant cells react to drought by

physiological modifications of the vacuole content and

osmotic pressure. In suspension cells there is a rapid

increase in vacuolar volume in response to salt stress

(Mimura et al. 2003). Pollen may be affected under water

stress but the vacuole does not seem to be the main case of

water injury during pollen development. The most sensitive

step of development is meiosis, upsets though pollen

remains sensitive through development (Saini 1997). In

fact, water stress upsets carbohydrate metabolism in the

whole anther and inhibits key enzymes of sugar metabo-

lism. Developing microspores/pollen grains are thus

deprived of nutrients, which affects their vigour and ability

to germinate.

In ripe pollen, vacuoles are reduced in size when pres-

ent. Pollen undergoes stress, especially during presentation

and dispersal, but the role of vacuoles has not been clearly

demonstrated. Depending on the capacity of pollen to

retain water, the following categories can be identified:

(i) pollen not dehydrated when anther opens and dispersed

in the sea (McConchie and Knox 1989); (ii) pollen that has

completed the process of dehydration prior to anther

opening; (iii) pollen that has not completed the process of

dehydration when the anther opens but completes it during

exposure or dispersal; (iv) pollen that is not dehydrated

when the anther opens and has no mechanism for retaining

water (Pacini 2000). Only type (i) and those of orchids

seem to have vacuoles at dispersal (McConchie and Knox

1989; Pacini and Hesse 2002).

Vacuoles and cell polarisation

Before the discovery that the cytoskeleton is responsible

for cyclosis and movement of the nucleus and organelles,

the formation of a big vacuole in late microspore stage was

interpreted as a means for nuclear displacement and

asymmetric mitosis (Knox and Ducker 1991). This does

not seem completely true because extensive vacuolisation

may occur at different stages, either earlier or later with

respect to pollen mitosis. Besides, in orchids extensive

vacuolisation does not occur but the microspore nucleus

polarizes before pollen mitosis.

On the other hand, the location of the asymmetric

division plane in the microspore also depends on the

location of microtubules close to the plasma membranes

222 Planta (2011) 234:217–227

123

(Twell et al. 1998). In species with huge vacuoles, the

position of the nucleus in the microspore before the first

pollen mitosis is therefore presumably due to the size of the

vacuole in the microspore and the position of the micro-

tubules generating the pre-prophase band. Analysis of

mutant Arabidopsis thaliana showed the crucial role of

microtubules orientation and the vacuole seemed to have a

secondary role (Twell et al. 1998).

Plastid function depends on information originating in

the nucleus. The extension of the vacuole in the microspore

after meiosis concentrates the organelles around the

nucleus favouring exchange between the nucleus and the

semi-autonomous organelles (Nagata et al. 2000).

Vacuolisation and pollen embryogenesis

Pollen embryogenesis is a biotechnological process leading

to the formation of double haploid plants from microsp-

ores, which would normally become pollen grains. The

process is characterised by a switch in microspore physi-

ology from a gametophytic programme to an embryogenic

sporophytic programme. The switch is triggered by stress.

The phase of microspore vacuolisation is the most

suitable time to collect microspores or early bicellular

pollen grain. In this stage, a group of specific so-called

‘‘early’’ genes are expressed in the microspore (Honys and

Twell 2003). Later on after vacuole regression in maturing

pollen it becomes almost impossible for the developing

gametophyte to change its developmental programme.

During vacuolisation the microspore is still plastic and can

take another development pathway, though the implica-

tions of the vacuole in this trait are still unclear.

Using the microspore as a tool for pollen embryogene-

sis, attention must be paid to osmotic potential during

microspore isolation and pre-treatment (Hoekstra et al.

1993; Jacquard et al. 2009). Most pre-treatment media are

liquid, which requires that the osmotic pressure of the

medium be properly adjusted. If the osmotic pressure of

the medium is lower than that of the vacuolar sap, the

microspores may burst by massive water uptake. Con-

versely, a high osmotic pressure in the pre-treatment

medium stresses the microspore and temporarily plasmo-

lyses it. This stress triggers microspore reorientation in

many cases (Wojnarowiez et al. 2004). Though the influ-

ence of the osmotic stress on microspore physiology is

unknown, vacuoles are the first target when inducing pollen

embryogenesis.

Observations of the vacuole by transmission electron

microscopy using the tonoplast feature as marker can be

used to determine microspore competence for embryo-

genesis at a very early stage of the process. Indeed, sen-

sitive microspores are characterised by deposition of a

tannin layer inside tonoplasts (Sangwan and Camefort

1983). The relation between the presence of tannin in

tonoplasts and microspore competence for haploid

embryogenesis is unclear.

The vacuole of the microspore is also affected during

the embryogenic process (Clement et al. 2005). To induce

pollen embryogenesis, microspore stressing pre-treatment

is done during microspore vacuolisation. The behaviour of

stressed microspores has been studied in detail using cell

tracking in tobacco, barley and wheat (Touraev et al. 1997;

Kumlehn and Lorz 1999; Indrianto et al. 2001). The

nucleus gradually moves to the centre of the microspore,

possibly guided by the cytoskeleton. At the same time, the

vacuole breaks up and sub-vacuoles become regularly

distributed around the central nucleus to form a typical

‘‘star like’’ microspore (Fig. 7). These events are regarded

as the first signs of microspore reorientation and are a

necessary step in the process of pollen embryogenesis

(Indrianto et al. 2001). The ‘‘star like microspore’’ devel-

ops an embryogenic programme. Maraschin et al. (2005)

demonstrated in barley that androgenesis is not only linked

to a peculiar type of vacuole, but also to the presence of

intine.

In the days after pre-treatment, organelle degradation and

lytic activity of the vacuole related to change in nuclear

ploidy can be observed (Sangwan and Sangwan-Norreel

1987; Garrido et al. 1995). This indicates that the pro-

gramme of cytoplasm reorganisation initiated in the

microspore in vivo after meiosis is maintained under in vitro

conditions, despite the drastic treatment of microspores.

Reisen et al. (2005) stressed the vacuole of suspension

culture cells, osmotically or with PEG, demonstrating an

increase in tonoplast size and ‘‘double’’ membrane struc-

ture. They postulated that this response creates a reserve

membrane for when original conditions are restored.

During embryogenesis the central vacuole does not

disappear, but divides simultaneously with the cell, pro-

viding one vacuole per cell at each stage of early

embryogenesis (Sangwan and Sangwan-Norreel 1987).

A similar situation occurs in somatic cells of carrot and

Cichorium during de-differentiation and embryogenesis

(Sangwan and Sangwan-Norreel 1987). This suggests

fragmentation of the central vacuole during these processes

and regression of the central vacuole during differentiation

and maturation. Tonoplasts seem to play an active but

unknown role during in vitro embryogenesis.

Molecular biology of pollen vacuoles

Rojo et al. (2003) showed that the protein complex local-

ised in tonoplasts is common to many vegetative cell types

and conserved by virtue of homology with yeast tonoplast

Planta (2011) 234:217–227 223

123

proteins. The role of vacuolar ATPase in male gameto-

phytes was revealed by Dettmer et al. (2005) and indeed a

large central vacuole where osmotically active substances

are transported is necessary for cell expansion. Besides,

plant cells and especially ripe pollen must maintain their

intracellular ion homeostasis throughout adverse conditions

that may occur during pollination. Several vacuolar ion

channels, such as H?-ATPase or calcium channels, appear

to be expressed at all stages of pollen development

(Jakobsen et al. 2005; Peiter et al. 2005; Bock et al. 2006)

and during germination and pollen tube growth (Pertl et al.

2009; Sheoran et al. 2009). Strompen et al. (2005) found

that vacuolar H? ATPase, was present during somatic

development but absent during development of both male

and female gametophytes. It was also shown that vacuolar

acidic invertase involved in wall metabolism and sugar

metabolism is up-regulated during pollen development and

tube growth (Holmes-Davis et al. 2005; Dai et al. 2006).

Plastid metabolism interferes with that of vacuoles.

Using mutants, Niewiadomski et al. (2005) demonstrated

that plastidial glucose 6 phosphate is essential for pollen

maturation and embryo sac development. Mutant pollen

does not form lipid bodies and pollen structure disinte-

grates when vacuole organisation is disrupted.

In recent years, the function of aquaporins, a class of

membrane proteins facilitating water diffusion across

membranes, has been characterised (Luu and Maurel

2005). The presence and/or significance of these molecules

has not been well studied during pollen ripening, presen-

tation and dispersal. They may have a role in anther and

pollen dehydration before dehiscence. They have been

detected in most anther tissues (Ruiter et al. 1997; O’Brien

et al. 2002; Alexandersson et al. 2005; Bots et al. 2005).

Some aquaporin genes are pollen specific (Bock et al.

2006; Maeshima and Ishikawa 2008; Soto et al. 2008),

suggesting that expression of aquaporins in pollen is under

strict regulation by the gametophytic programme. Although

they are likely involved in the process of water accumu-

lation within developing vacuoles during vacuolation, the

involvement of aquaporins has not yet been clearly

reported in pollen development but rather in pollen ger-

mination upon contact with the stigma (Ikeda et al. 1997).

Thus, their role is probably crucial when pollen is exposed

to the environment. We can suppose that the actual func-

tion of the aquaporins may be in the pollen tube elongation,

in the stigma-style transition and in the pollen rehydration.

Conclusions and perspectives

Structural and chemical research into pollen vacuoles are

both afflicted by methodological problems. Structural

observations are mainly done with material rehydrated

during fixation because glutaraldehyde and osmium

tetroxide are dissolved in water. This means that pollen

dispersed with low water content rehydrate during fixation.

We do not know if the small vacuoles sometimes present in

ripe pollen images are due to this rehydration. Even if we

assume that small vacuoles are part of pollen structure, it is

impossible to separate the vacuoles to analyse their

contents.

Vacuoles seem practically absent in ripe pollen, because

extensive translucent areas as in the vegetative part of the

plant, are not observed. Only small areas with electron

opaque deposits can be seen and are interpreted as phytate

deposits. They were not observed when pollen was col-

lected from open anthers or if the fixative was devoid of

water. However, the cytoplasm was full of small vesicles

containing polysaccharides content (Weber 1988; Franchi

et al. 1996; Yamamoto et al. 2003) and the cytosol con-

tained sucrose, glucose, fructose (Speranza et al. 1997) and

lipid deposits of spherosoms, but these can be considered

small vacuoles created by ER dilation.

In this respect, mature pollen is similar to seeds: con-

ventional vacuoles are absent from endosperm and embryo

cells of mature seeds. Though ripe seed vacuoles differ

from those of pollen because they store proteins (Marty

1999), in both pollen and seeds water content dramatically

decreases before dispersal, with some exceptions such as

PHP and recalcitrant seeds (Baskin and Baskin 1998).

Fig. 7 Dynamics of vacuoles in

the early steps of microspore

embryogenesis

224 Planta (2011) 234:217–227

123

Partially dehydrated pollen (Nepi et al. 2001; Franchi et al.

2002) and orthodox seeds (Baskin and Baskin 1998) resist

to severe environmental stresses better. The behaviour of

male and female gametophytes is different because the

latter always have a big central vacuole (Howden et al.

1998).

The behaviour of vacuoles is not uniform throughout the

angiosperms during pollen development. There seem to be

two main types: extensive vacuolisation during develop-

ment and its virtual absence in ripe pollen, as in almost all

angiosperms; reduced vacuolisation during development

and presence even in the ripe stage as in orchids. This may

be because pollen of orchids with pollinia as pollen dis-

persal unit is PHP, not totally exposed to the environment,

but protected inside the anther until pollinator arrival

(Pacini and Hesse 2002, 2004). In fact, it stays inside the

anther until loaded by insects (Pacini and Hesse 2002).

Under such sheltered conditions even pollen dehydration is

not necessary and pollen vacuoles are present, and resist

well out of the anther because of their high sucrose content

(Nepi et al. 2001; Franchi et al. 2002).

If vacuoles as water reserves are absent in ripe pollen,

even in PHP, new vacuoles are formed during rehydration

and early germination (Southworth and Dickinson 1981;

Hicks et al. 2004). The content of reserve containing ves-

icles is mobilised for activation and germination. There is

little data on this topic, despite its great interest, especially

if structural observations are linked or proceed with

molecular biology observations on gene action.

In the last decade, many genes have been shown to be

specifically expressed at special steps of pollen develop-

ment that coincide with the formation or degradation of

vacuoles in the microspore or pollen grain (Oldenhof et al.

1996). Unfortunately the physiological function of many of

these genes is unknown (Honys and Twell 2003) despite

correlations with vacuolisation.

Several pathways remain to be investigated related to

pollen vacuole and its role in pollen biology/biotechnology.

Regarding pollen behaviour, it would be of interest to

determine the role of vacuole in pollen tolerance to dehy-

dration during pollination. Pollen survival is a key step of

sexual reproduction and its capacity to tolerate water stress

conditions the achievement of the reproductive process.

Furthermore, small vacuoles in mature pollen grain are

likely involved in the process of pollen rehydration pre-

ceding pollen tube germination. There is currently only

poor information on this process. Regarding pollen

embryogenesis, vacuole in microspore/pollen at the time of

pre-treatment plays a central role since it conditions the

position of the nucleus which determines the symmetry of

the first division following microspore/pollen reorientation.

Here again, data are missing about mechanisms regulating

this process.

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