Annals of Botany 109: 1201– 1213, 2012
doi:10.1093/aob/mcs070, available online at www.aob.oxfordjournals.org
INVITED REVIEW
Water status and associated processes mark critical stages in pollen development
and functioning
Nurit Firon1,*, Massimo Nepi2 and Ettore Pacini2
1
Institute of Plant Sciences, The Volcani Center, ARO, Bet Dagan 50250, Israel and 2Department of Environmental Sciences,
Siena University, 53100 Siena, Italy
* For correspondence. E-mail [email protected]
Received: 21 December 2011 Returned for revision: 10 February 2012 Accepted: 24 February 2012 Published electronically: 19 April 2012
† Background The male gametophyte developmental programme can be divided into five phases which differ in
relation to the environment and pollen hydration state: (1) pollen develops inside the anther immersed in locular
fluid, which conveys substances from the mother plant – the microsporogenesis phase; (2) locular fluid
disappears by reabsorption and/or evaporation before the anther opens and the maturing pollen grains undergo
dehydration – the dehydration phase; (3) the anther opens and pollen may be dispersed immediately, or be
held by, for example, pollenkitt (as occurs in almost all entomophilous species) for later dispersion – the presentation phase; (4) pollen is dispersed by different agents, remaining exposed to the environment for different
periods – the dispersal phase; and (5) pollen lands on a stigma and, in the case of a compatible stigma and
suitable conditions, undergoes rehydration and starts germination – the pollen–stigma interaction phase.
† Scope This review highlights the issue of pollen water status and indicates the various mechanisms used by
pollen grains during their five developmental phases to adjust to changes in water content and maintain internal
stability.
† Conclusions Pollen water status is co-ordinated through structural, physiological and molecular mechanisms.
The structural components participating in regulation of the pollen water level, during both dehydration and
rehydration, include the exine (the outer wall of the pollen grain) and the vacuole. Recent data suggest the
involvement of water channels in pollen water transport and the existence of several molecular mechanisms
for pollen osmoregulation and to protect cellular components ( proteins and membranes) under water stress. It
is suggested that pollen grains will use these mechanisms, which have a developmental role, to cope with
environmental stress conditions.
Key words: Pollen, water status, dehydration, rehydration, angiosperm pollen, pollination.
IN T RO DU C T IO N
Organisms, and their cells, require mechanisms to maintain internal stability in the face of developmental and environmental
changes. The American physiologist Walter Cannon (1871 –
1945) termed this ability ‘homeostasis’ (homeo means ‘the
same’ and stasis means ‘standing or staying’). Homeostasis
may thus be defined as the ability of an organism or cell to maintain internal equilibrium by adjusting its physiological processes. In this review, we pinpoint five phases during male
gametophyte development and functioning that require maintenance of specific water levels, via the use of various structural,
physiological and molecular mechanisms. These five phases
differ in the optimum water/hydration levels needed to suit specific biological functions, as detailed further on. The significance of the existence of potential ‘water homeostasis control
points’ will also be addressed in relation to maintaining pollen
quality and function upon exposure to environmental stresses.
Changes in pollen volume and water content during
the developmental programme
The male gametophyte developmental programme can be
divided into five phases which differ in relation to the
environment and pollen hydration state. (1) Pollen develops
inside the anther immersed in locular fluid, which conveys
substances from the mother plant – the microsporogenesis
phase. (2) The locular fluid disappears by reabsorption and/
or evaporation before the anther opens (Pacini et al., 2006,
and references therein) and the maturing pollen grains
undergo dehydration – the dehydration phase. (3) The anther
opens: pollen may be dispersed immediately, or be held for
later dispersal by, for example, pollenkitt, as occurs in
almost all entomophilous species (Pacini et al., 2006, and
references therein) – the presentation phase. (4) Pollen is dispersed by different agents, remaining exposed to the environment for different periods – the dispersal phase. (5) Following
dispersal, pollen will land on a stigma and, in the case of compatible stigma and suitable conditions, will undergo rehydration and start germination – the pollen– stigma interaction
phase (Fig. 1).
Pollen volume starts increasing during microspore wall
formation and continues to increase until dehydration
(Pacini, 1994). The increase in volume parallels an increase
in water content and vacuolation, part of the space being
occupied by vacuoles (Fig. 1, phase 1). At the start of the
dehydration phase ( phase 2), pollen water content starts to
decrease, reaching a minimum at maturity. This causes a
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Firon et al. — Pollen water status, development and functioning
73
Pollen tube
emission
61
72
Meiosis
H2O
1
2
3
4
Vacuolation
Stigma
5
71
1st haploid
mitosis
Microspore
release
8
9
6
7
Anther
opening
Pollination
Dehydration
Phase 1
Phase 2
Microsporogenesis
Maturation &
dehydration
Rehydration
3
Phase 4
Presentation &
dispersal
Phase 5
Pollen–stigma
Interaction &
rehydration
F I G . 1. A semi-diagrammatic scheme of male gametophyte development from the tetrad stage to pollen tube emission, indicating changes in volume (solid line)
and water content (dashed line). The scheme does not consider species with tricellular pollen. Vacuoles are in blue, starch is in red and cytoplasmic carbohydrates
are in pink. (1) Late tetrad stage. (2) Early microspore stage; small vacuoles are formed. (3) Late vacuolated microspore stage, before the first haploid mitosis;
small vacuoles merge into a large one. (4) Early bicellular stage; starch deposition begins, as well as vacuole reduction. (5) Late bicellular stage; vacuoles are
absent, starch deposition reaches maximum levels. From stage 5, two possibilities are illustrated (6 and 61). (6) Almost mature pollen grain; starch is completely
hydrolysed and carbohydrates are present in the cytoplasm. (61) Almost mature pollen grain; starch is only partially hydrolysed. (7) Mature pollen, of the partially
hydrated type (recalcitrant; Nepi et al., 2001), containing cytoplasmic carbohydrates. (71) Mature pollen of the partially dehydrated type (orthodox; Nepi et al.,
2001), containing cytoplasmic carbohydrates. (72) Mature recalcitrant pollen with starchy carbohydrate reserves. (73) Mature orthodox pollen with starchy carbohydrate reserves. (8) After landing on the stigma, pollen, independently of the hydration state and type of carbohydrate reserves, rehydrates and small vacuoles are
formed. (9) Small vacuoles merge into a large one and a pollen tube is emitted. Starch and cytoplasmic carbohydrates interconvert during presentation and dispersal, enabling control of water loss and water influx, and are consumed (or transformed) during rehydration and germination. Pollen water content (dependent
on water absorbed from the anther through the tapetal cells and then through the loculus) starts increasing at the microspore stage, in parallel with the initial
increase in volume, up to a few days before flower opening (according to the species; phase 1). Upon beginning the dehydration phase (phase 2), pollen
water content starts to decrease, reaching a minimum at maturity. During presentation (phase 3) and dispersal ( phase 4), water content adjusts to environmental
changes (depending on the species) and will increase sharply during rehydration, upon landing on a compatible stigma ( phase 5). This precedes the emission of
the pollen tube which grows in the style and will reach the ovule where sperm cells will be delivered for fertilization. It should be noted that the illustrated changes
in pollen water content during development are speculative since, to the best of our knowledge, no such measurements have ever been reported. The time scale for
water content during the different phases varies widely according to the species and is deeply affected by the environment during dispersal. In general: phase 1 is
the longest, lasting from several days to several months, whilst the other phases last from seconds to days. Pollen volume starts increasing significantly at the early
microspore stage and continues to increase until partial dehydration. Pollen volume decreases during anther and pollen dehydration and, similar to water content,
adapts to environmental conditions during dispersal, increasing during rehydration.
decrease in volume, in most cases with a change in pollen
shape (Nepi et al., 2001). During presentation and dispersal
( phases 3 and 4), water content, though at a low (or even
minimal) level, adapts to environmental changes, and will increase sharply during rehydration, upon landing on a compatible stigma ( phase 5). Pollen volume may change during
dispersal as well, depending on the relative humidity (RH)
of the environment (Lisci et al., 1994; Nepi et al., 2001,
and references therein). When pollen lands on a compatible
stigma, it rehydrates and germinates (Heslop-Harrison,
1987). Wodehouse (1935) termed the changes in pollen
volume and shape during dehydration (illustrated in Fig. 2),
dispersal and rehydration ‘harmomegathy’; these changes
create mechanical stress which must be sustained by the
pollen walls, plasma membrane and protoplast (Blackmore
and Barnes, 1986).
Firon et al. — Pollen water status, development and functioning
1203
WAT E R S TAT E AN D C H A R AC T E R I S T I C S
Water has several roles in biological systems: it regulates biological reactions, serves as a fluid medium and stabilizes
macromolecular structure. Therefore, removal of water from
biological tissues affects their ability to function and may
induce deleterious effects (Alpert and Oliver, 2002).
Desiccation-tolerant organisms, such as seeds and pollen, are
capable of surviving the removal of their cellular water and
may also be able to survive in a dry state for extended
periods of time (Alpert and Oliver, 2002). In 1986, Burke
hypothesized that the seed cytoplasm can enter into a glassy
state (Buitink and Leprince, 2004, and references therein).
Glass is a thermodynamic liquid which can be defined as a
supercooled liquid with an extremely high viscosity (Buitink
and Leprince, 2004, and references therein). Intracellular
glass, which exhibits slow molecular mobility and high molecular packing, has recently been suggested to be essential
for the storage stability of seeds (Buitink and Leprince,
2008). Non-reducing sugars, such as sucrose, are likely to
play multifunctional roles during the loss of cellular water.
At high water contents, early on in the drying stage, they
might act as compatible solutes. Through preferential exclusion, protein unfolding is avoided and membrane disturbance
is restricted. Upon further water loss, sugar molecules can
replace water at hydrogen-bonding sites to preserve the
native protein structure and spacing between phospholipids
(Buitink and Leprince, 2004, and references therein). Potts
(2001) stressed the importance of both sucrose and trehalose
in replacing water around the polar head groups of membranes
and in forming glasses in the dry state. Due to their abundance
in the cytoplasm, proteins have also been suggested to play a
role in glass formation. During maturation, seeds and pollen
accumulate both non-reducing sugars and late embryogenesis
abundant (LEA) proteins (this issue is further elaborated
upon below), and it was therefore suggested that the two
types of molecules interact in the formation of a glassy state
(Buitink and Leprince, 2004, and references therein).
F I G . 2. Cryo-scanning electron micrograph of Petunia hybrida pollen after
anther opening. (A) Pollen before flower opening. (B) Pollen after flower
opening. Pollen grains lose water after flower opening and change in size
and shape, from spheroid to ovoid. The furrow area is distended in the
closed flower (asterisks) whereas it is folded in the open flower after partial
pollen dehydration (arrows). Pictures were taken at the Department of Plant
Cytology and Morphology, University of Wageningen, The Netherlands, in
1999 using a JEOL 6300F field emission cryo-SEM.
The aim of this review is to highlight the issue of pollen
water status and point out the various mechanisms used by
pollen grains during the five phases of their developmental
programme to adjust to changes in water content and maintain internal stability. In addition, we argue that pollen
grains may also use these mechanisms for coping with
stressful environmental conditions. It should be noted
that direct data with regard to water content in the anther
and pollen are very limited. The review will thus focus
on the available data, and relate them to mechanisms
used in other plant systems/tissues, highlighting potential
homeostasis control points and discussing future research
avenues.
M IC RO S PO ROG E NE S IS , M AT U R AT IO N
A ND DE H Y DR AT IO N ( P HA S E S 1 AN D 2 )
Pollen grain volume decreases slightly after meiotic cleavage
and starts to increase during exine and intine formation (constituting the two walls of the pollen grain); with dehydration,
the volume decreases again (Pacini, 1990). Pollen and spores
are the only plant cells delimited by two walls with different
structures, compositions and functions. These walls are
formed centripetally – first the elastic and flexible external
exine, and then the internal intine. Exine is lacking, discontinuous or extremely reduced in a small number of plant
groups, e.g. seagrasses and certain tropical monocots such as
Musaceae, Lauraceae and Zingiberaceae living in environments with high RH (Pacini, 1994, and references therein).
Pollen walls are subject to mechanical stress, especially at
the apertures, during anther and stigma hydration. During dehydration, the pollen wall folds in on itself to prevent further
desiccation (Fig. 2). The structural patterning of the pollen
wall – an axially elongated aperture of high compliance and
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Firon et al. — Pollen water status, development and functioning
intricate sculpturing – is critical for predictable and reversible
folding patterns (Katifori et al., 2010).
Dehydration causes both the anther to dehisce and the
pollen grain to become ‘dormant’ (in analogy with seeds),
i.e. its biochemical processes function at a low rate. In
species without exine, such as seagrasses and some
Musaceae, dehydration is reduced or absent (Franchi et al.,
2011, and references therein). This emphasizes the importance
of the exine in accommodating changes in pollen volume due
to uptake and loss of water.
Exine function
Bowman et al. (2007) and Morant et al. (2007) have suggested that a key innovation in the first land plants colonizing
the terrestrial environment 600 – 450 million years ago was acquisition of the ability to generate the sporopollenin polymer
to protect haploid spores. Functional and biochemical roles
of the exine have been studied using mutants defective in
pollen wall development and exine deposition, such as defective in exine formation1 (dex1), ms2, no exine formation1, faceless pollen1 and cer6, as well as the recently characterized
acos5 and cyp704B2 mutants (Aarts et al., 1997; Fiebig
et al., 2000; Paxson-Sowders et al., 2001; Ariizumi et al.,
2003; Kunst and Samuels, 2003; Souza et al., 2009; Li
et al., 2010). Jung et al. (2006), for example, characterized a
T-DNA insertional mutant in the Wax-deficient anther1
(Wda1) gene of rice (Oryza sativa) which shows significant
defects in the biosynthesis of very-long-chain fatty acids in
the anther layers. This gene was found to be strongly expressed
in the epidermal cells of anthers. Scanning electron microscopy analyses of cells with this mutation showed that
epicuticular wax crystals are absent in the outer layer of the
anther. The authors reported that the wda1 anthers were
severely shrunken by dehydration and easily damaged by
physical force, and that microspore development was severely
retarded and finally disrupted as a result of defective pollen
exine formation in the mutant anthers.
Pollen vacuole function
The plant vacuole acts as a storage compartment. It has roles
in turgor regulation, cell signalling and degradation, and it has
been shown to accommodate high flux rates of water and small
solutes across its membrane (‘tonoplast’; Kaldenhoff and
Fischer, 2006), suggesting an important function under conditions of stress (Li et al., 2008). Vacuolation usually occurs
from the tetrad stage of pollen development onward (Pacini,
1994). Two vacuolation events occur in Lycopersicum peruvianum and Prunus avium, among many other plants, the
first during the microspore stage and the second during
the bicellular stage. Only one vacuolation occurs, during the
microspore stage, in monocots such as Lolium perenne
(Pacini, 1994). Vacuolation in developing pollen seems to
follow pollen volume changes as well as amylogenesis
(Pacini et al., 2011). Vacuoles may have important functions
during dehydration and subsequent rehydration on the
stigma, similar to other plant systems where tonoplast water
channels (aquaporins) have been shown to play an important
role in maintaining water balance under changing environmental conditions (Kaldenhoff and Fischer, 2006).
Uniqueness of the loculus and the locular fluid
The loculus, surrounding and serving as the immediate environment for the developing pollen grains, is formed during
meiotic prophase, owing to enlargement of the anther and detachment and separation of meiocytes (Pacini and Franchi,
1983). The dimension of the loculus increases up to pollen
grain maturity. It is not known, however, whether the increase
in volume of the locular fluid is proportional to the increase in
volume of the developing pollen. As reported for Smilax
aspera L. plants, during the initial stages of loculus formation,
it stores substances containing polysaccharides which can be
stained by PAS ( periodic acid – Schiff ) (Pacini and Franchi,
1983). In Lilium, the locular fluid was shown to store
sucrose, glucose and fructose during pollen development
(Castro and Clément, 2007) and was suggested to serve as a
transitory site for sugar storage. There are different forms of
loculi depending on the type of tapetum, pollen dispersal
unit and pollen grain size (Pacini, 2010). The presence of
locular fluid, which separates the microspores from the cells
of the mother plant during a significant period of their
development, may contribute to the high sensitivity of the
developing pollen grains to the environment.
Roles of the filament and anther transpiration
At the level of the anther, the mechanism leading to dehydration varies from one taxonomic group to the next. It is
greatly influenced by environmental factors such as rain, humidity and sunlight, as well as variations in RH with time of
day. Dehydration may occur in two different, but not mutually
exclusive, ways: active water resorption by other flower organs
as in Petunia, or desiccation by passive transpiration as in
Tradescantia (Pacini and Franchi, 1984). Most of the
anther’s water is transported to the expanding filament in
Lilium longiflorum, and anther dehiscence is delayed if the
filament is tied (Heslop-Harrison et al., 1987). Mechanisms
facilitating the evaporation of water are an abscising tissue
in the filament hindering liquid inflow, and discontinuties in
the anther cuticle, facilitating transpiration (Pacini, 1994, and
references therein). Pollen volume decreases due to loss of
water, and pollen water content is in equilibrium with the environment during dispersal. The water content of ripe pollen,
i.e. when it has reached equilibrium with the environment,
generally ranges from 20 to 50 % (Pacini, 1994, and references
therein).
Variations in water and carbohydrate content in mature pollen
It should be noted that, as reported for mature seeds (Hay
et al., 2010), mature pollen grains derived from the same
mother plant, or even from the same flower, are not necessarily
uniform. Pollen grains may differ in their water and carbohydrate contents, reflecting the pollen’s position inside the
loculus and inside the flower, and the consequent effects of
the environmental parameters. These variations may be inherent, due to a microenvironment effect of the mother plant
Firon et al. — Pollen water status, development and functioning
1205
Water content :
43 %
13 %
8·3 %
Viability :
85 %
81 %
13 %
DEHYDRATION at low relative humidity
F I G . 3. Cryo-fractures of Cucurbita pepo pollen observed with a cryo-SEM after freeze-drying (10 min at –90 8C in a vacuum of 5 × 1028 hPa). The sites
containing water appear as vesicle-like structures (dark holes) in the cytoplasm. These vesicles are highly abundant in the cytoplasm of C. pepo pollen from
just-opened anthers (water content 43 %). They decrease in pollen dehydrated to 13 % water content and they are no longer evident in pollen dehydrated to
8.5 % water content. Amyloplasts (a) and their imprints (asterisks) are evident in C. pepo pollen. Pollen viability is high at both 43 and 13 % water content,
but it decreases drastically upon further dehydration to 8.5 % water content. This degree of dehydration is induced by pollen exposure to 30 % relative humidity
for 90 min (see data in Nepi et al., 2010). Pictures were taken at the Department of Plant Cytology and Morphology, University of Wageningen, The Netherlands,
in 1999 using a JEOL 6300F field emission cryo-SEM. Scale bar ¼ 2 mm.
during pollen development, e.g. nourishment through the
tapetum layer. Examples of different types of microspore –
tapetum interactions are presented by Pacini (2010).
P R E S E N TAT I O N ( P H A S E 3 )
Anther opening
Anther dehiscence involves anther wall rupture at the stomium
to allow pollen presentation and release. It is a complex
process requiring proper cell differentiation and dehydration.
It is agreed that as a general rule, low RH accelerates anther
opening, whereas high RH delays or inhibits the process
(Linskens and Cresti, 1988; Yates and Sparks, 1993; Lisci
et al., 1994). Explosive anther dehiscence in Ricinus communis L. (castor bean) was shown to be influenced by RH
(Bianchini and Pacini, 1996), with values .30 % inhibiting
anther opening and decreasing the distance the pollen is
launched, and a value of 98 % completely preventing anther
opening (Bianchini and Pacini, 1996). Matsui et al. (2000)
described a mechanism of anther dehiscence related to
pollen grain swelling in barley (Hordeum distichum L. emed.
Lam.), suggesting the involvement of potassium ions in the
swelling. In Allium triquetrum, anther opening occurred at a
specific moment of anther development and seemed to be
related to dehydration caused by reabsorption of water by contiguous tissues (Carrizo Garcia et al., 2006).
Pollen hydration status
Pollen hydration status at presentation is dependent on its developmental programme and is affected by anther dehydration as
well as by the environmental conditions encountered during
presentation. Two modes of pollen presentation have been
defined: ‘primary pollen presentation’ (Leins, 2000) indicating
pollen presented by the anther, and ‘secondary pollen presentation’ indicating pollen adherence to other parts of the flower
(Howell et al., 1993; Yeo, 1993; Ladd, 1994; Erbar and Leins,
1995) by virtue of pollenkitt (Pacini, 1996) or other devices
(Erbar and Leins, 1995, and references therein). Pollen hydration
status is its water content, which may change at anther opening,
during presentation (Lisci et al., 1994) or during dispersal. Nepi
et al. (2001) defined ‘partially dehydrated pollen’ (PDP) as
having ,30 % water content, whereas pollen containing .30
% water was termed ‘partially hydrated pollen’ (PHP). By
analogy with seeds, PDP has also been defined as orthodox or
desiccation tolerant (Tweddle et al., 2003), whereas pollen
with a higher water content is defined as recalcitrant or desiccation sensitive (Pacini et al., 2006; Franchi et al., 2011). In this
review, we use the terms orthodox and recalcitrant for
desiccation-tolerant and -sensitive pollen, respectively.
Recalcitrant pollen is almost ready for dispersal when the
locular fluid has disappeared. Orthodox pollen must lose water
to slow down its metabolism before it is ready for dispersal
(Pacini, 1990; Franchi et al., 2002). In Petunia hybrida ( possessing orthodox pollen), for example, pollen is still hydrated and
spherical when the anther opens and the flower is still closed;
pollen dehydration occurs after flower opening (Fig. 2;
M. Nepi, pers. obs.). During this process the orthodox pollen,
with three pores and/or furrows, for example, changes shape
from spherical when hydrated, to ovoid when partially dehydrated (Fig. 2). On the other hand, recalcitrant pollen is
usually devoid of furrows and generally spherical in shape,
with many, one or no pores (Franchi et al., 2002, 2011); it
does not change shape when water is lost, but its volume and
diameter decrease (Nepi and Pacini, 1993). However, if the
exine and/or intine are thin, they eventually collapse, as commonly seen in grass pollen (Heslop-Harrison, 1979). Most recalcitrant pollen lacks mechanisms for retaining water. If it does not
reach a compatible stigma quickly, it rapidly dehydrates and dies
(Fig. 3). The molecular mechanisms involved in pollen water
homeostasis include water channels that may facilitate water
movement, on one hand, and mechanisms that enable it to
retain water such as the accumulation of specific sugars and proteins, on the other hand (see details in the section ‘Molecular
mechanisms involved in enabling changes in pollen water
status’ below).
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Firon et al. — Pollen water status, development and functioning
Irrespective of pollen hydration status, when anther or
pollen dehydration is delayed (naturally or experimentally),
pollen of some species will start to emit a pollen tube in the
closed anther (Pacini and Franchi, 1982).
Success of orthodox pollen during presentation and the subsequent dispersal phase may be determined by: (a) the ability
to keep water content as constant as possible; (b) the ability to
adjust turgor pressure with changes in environmental parameters; and (c) the existence of molecular mechanisms that
will aid in maintaining (a) and (b).
Ecophysiology
When pollen is dispersed immediately upon anther opening,
its flight may be very short – a few centimetres at most and
lasting only a few seconds, as in the case of annual grasses.
If pollen is retained in the anther by pollenkitt or other
means, it may be exposed for periods ranging from a few
hours to several weeks, as in orchids, where dispersal
depends on pollinator availability (Neiland and Wilcock,
1995). In the case of Cucurbita pepo (a monoecious entomophilous species), the male flower opens at 0600 h and
closes at 1100 h (Nepi and Pacini, 1993); uncollected pollen
is not exposed again. This may be due to the fact that
Cucurbita has recalcitrant pollen which is vulnerable to
water loss, even during brief exposure. Corolla closure at the
end of anthesis is not limited to Cucurbita: it seems to be
typical of species with recalcitrant pollen, such as
Convolvulus sp., Mirabilis sp. and Hibiscus sp. (Franchi
et al., 2002), and to offer a way of avoiding dispersal of
pollen with low viability.
Pollen developmental arrest was demonstrated in hazel,
reflecting the dormancy period (Frenguelli et al., 1997). In
this case, pollen development lasted for several months (beginning 7 – 8 months before flowering in late winter or early
spring) and variations in pollen volume reflected the beginning
and end of the dormancy period (decreased and maximal
volume, respectively; Frenguelli et al., 1997).
In the case of Helleborus foetidus and H. bocconei, which
have orthodox pollen, the flower may last as long as 40 d,
but only a few anthers expose pollen each day (Vesprini
et al., 2002). However, the pollen maintains high viability
during exposure (lasting for 2 d), despite the fact that night
temperatures may fall below 0 8C, with day temperatures of
18 –20 8C during the blooming period. This suggests the existence of biochemical mechanisms that keep its viability high
with time, by avoiding water loss, water uptake and formation
of ice crystals in the cytoplasm (Vesprini et al., 2002), presumably via changes in the relative proportions of soluble carbohydrates. Experiments with Petunia hybrida, Trachycarpus
fortunei and Helleborus sp. demonstrated that when water
availability decreases – because of low RH which causes dehydration or because of low temperature which induces ice
crystal formation – the immediate response is an increase in
sucrose (Vesprini et al., 2002; Guarnieri et al., 2006; Nepi
et al., 2010). It is also interesting to note that an initial increase
in sucrose due to lower water availability is followed by restoration of the quantity of this sugar (M. Nepi, pers. obs.). This
suggests that some form of homeostasis maintains a constant
sucrose pool following a disturbance.
DISPERSAL (PHASE 4)
Pollen may leave the anther as single grains or en masse (Knox
and McConchie, 1986), in units consisting of different
numbers of grains. The means for holding the grains together
include: (a) common walls; (b) viscous fluids derived from the
tapetum or other parts of the anther; or (c) tangling of the
grains themselves as in certain marine monocots, or by
means of exine filaments or filaments derived from the
anther (Pacini, 2000).
During dispersal, pollen grains are exposed to the environment and need to withstand variable conditions of temperature
and RH. It has been shown for both seeds and pollen that
several physical and chemical changes may occur with time,
including decreased activities of enzymes, lipid peroxidation
and de-esterification, and Maillard reactions (Buitink et al.,
1998, and references therein). It is assumed that the formation
of highly viscous intracellular glasses decreases molecular mobility and impedes diffusion within the cytoplasm, thus
slowing the deleterious reactions and changes in structure
during ageing (Buitink et al., 1998, and references therein)
as indicated above in ‘Water state and characteristics’. In the
case of pea (Pisum sativum L.) and cattail (Typha latifolia L.)
seeds, for example, molecular mobility was found to be
inversely correlated with storage stability. Recently, using
three species of seeds with different dry matter reserves
(elm, safflower and maize with moisture contents ranging
from 0.00 to 0.15 g H2O per g dry weight), Zhang et al.
(2010) showed that molecular mobility decreases and then
increases with seed drying in a pattern resembling that of
seed ageing kinetics, thus confirming previous data (Buitink
et al., 1998). Aylor (2003) claimed that the relative water
content of corn pollen plays an important role in both its viability and flight dynamics in the atmosphere. A model was
created for the dynamics of water loss from pollen under a
variety of temperature and RH conditions, to predict effective
pollen transport distances (Aylor, 2003; Nepi et al., 2010).
In the case of recalcitrant pollen, there is generally a threshold value for pollen water content below which there is an
abrupt decrease of pollen viability. For C. pepo and Zea
mays, the threshold water content is 13 and 28 %, respectively
(Fonseca and Westgate, 2005; Nepi et al., 2010).
Because desiccation tolerance is closely related to pollen
viability and longevity, it has a strong influence on reproductive strategy and pollination (Dafni and Firmage, 2000).
Species with orthodox pollen may wait a long time for pollination to occur. In contrast, species with recalcitrant pollen rely
on rapid transport of their pollen to a compatible stigma, so
that it can germinate and fertilize the ovules (Nepi et al.,
2010). The issue of pollen and seed desiccation tolerance in
relation to dispersal and survival is further elaborated upon
by Franchi et al. (2011).
PO L L E N – S T I G M A IN T E R ACT I O N ( P H A S E 5 )
This phase begins when the pollen grain lands on and adheres
to the stigma of a flower. If conditions are suitable, the pollen
grain germinates to produce a pollen tube. The pollen tube
invades the stigma and grows through the style toward the
ovary, where it enters an ovule, penetrates the embryo sac
Firon et al. — Pollen water status, development and functioning
and releases two sperm cells, one of which fertilizes the egg,
while the other fuses with the two polar nuclei of the central
cell to form the triploid endosperm. Pollen – pistil interactions
involve a continuous exchange of signals between the pollen
and the maternal tissue of the pistil (Hiscock and Allen,
2008). Heslop-Harrison (2000) described these events as
‘gates opening’, creating the microenvironment that will
enable pollen germination, and illustrating the sequence of
events that take place as the female tissues of the stigma,
style and ovary mature and prepare for pollination and fertilization. An interesting example is given by Heslop-Harrison
(2000) to describe a particular ‘gate opening’ sequence in
the case of Trifolium pretense: at maturity, the stigma head secretion has, among other constituents, a sucrose concentration
of approx. 35 %, the optimum for pollen hydration and germination, but not for continued tube growth; sucrose concentration in the stylar fluid drops to approx. 12– 15 %, the
optimum for tube growth but not for germination.
Pollen adhesion occurs only in angiosperms and is a prerequisite for pollen hydration; the opposite holds true for gymnosperms (Nepi et al., 2009). The environment of the landing
site determines physical recognition, which enables proper hydration to take place. The increasing percentage of pollen
volume from the dry to hydrated state varies between species
and depends on the hydration site and the pollen grain itself
(Pacini, 1990). The internal factors are: hydration of the
intine, selective permeability of the plasma membrane, hydration state of the cytoplasm and the accommodation capacity of
the walls. In grasses, fresh mature pollen grains germinate very
rapidly on suitably receptive stigmas, with germination in the
small-grain cereals taking place in ,5 min, and within 1 min
for Triticum aestivum ‘Sonora’ (reviewed by Heslop-Harrison,
1979). The rapid transition of the male gametophyte from a
state of comparative inactivity in the grain to one of vigorous
growth implies a rapid re-establishment of normal metabolism,
including the synthetic capacity required to build the pollen
tube wall. Activation of the pollen grain depends on rehydration, and this is dependent on the inflow of water from the
stigma after attachment of the grain. The speed with which
an appropriate water balance is reached will depend on both
the capacity of the stigma to support the flow and the requirements of the pollen grain. For mature, germinable pollen, the
period from capture to tube emergence is affected by the
degree of hydration of the grains at the moment of capture
(Heslop-Harrison, 1979). In pistachio (recalcitrant), pollen
stored at room temperature only germinates after pre-hydration
(Vaknin and Eisikowitch, 2000).
Pollen hydration takes place prior to emission of the pollen
tube. A nice example is given by Heslop-Harrison (1979) for
rye (Secale cereale L.) pollen, demonstrating water exudation
from the aperture before the beginning of tube tip growth; in
this particular case, germination took place 2 min and 20 s
after contact.
The stigma
Angiosperm stigmas can be classified into two broad categories, wet and dry, depending on whether they possess
surface secretions (Heslop-Harrison and Shivanna, 1977;
Heslop-Harrison, 1981). As summarized by Edlund et al.
1207
(2004), water immediately surrounds grains that land on a
wet stigma, but those that land on a dry stigma mobilize
their lipid-rich pollen coat to form an interface between the
two cell surfaces. This interface is converted into a histochemically distinguishable form thought to promote water
flow (Elleman and Dickinson, 1986; Elleman et al., 1992).
Water, nutrients and other small molecules are transported
rapidly into the grain from the stigma exudate (wet stigmas)
or stigma papillae (dry stigmas) by mechanisms that remain
unclear. The involvement of water channels in the rapid and
regulated water release from the stigma to the pollen has
been suggested (Dixit et al., 2001; Swanson et al., 2005). In
addition, the stigma cuticle may be uniquely permeable to
water traffic (Lolle et al., 1997, 1998; Pruitt et al., 2000).
Heslop-Harrison and Heslop-Harrison (1980) showed that the
cuticle of maize silk is discontinuous, allowing quick rehydration. Regardless of the transfer mechanism, pollen hydration is
often regulated both temporally and spatially. Inappropriate or
delayed hydration can have disastrous consequences, leading
to premature germination within the anther (Pacini and
Franchi, 1982; Johnson and McCormick, 2001) or germination
on the wrong surface (Lolle and Cheung, 1993; Lolle et al.,
1998).
The outer epidermal cell wall and cuticle present a semipermeable barrier that maintains the external integrity of the
plant and regulates the passage of various classes of molecules
into and out of the organism. During vegetative development,
the epidermal cells remain relatively inert. During reproductive
development and fertilization, however, the epidermis is developmentally more labile and participates in two types of
contact-mediated cell interactions: organ fusion and pollen hydration (Pruitt et al., 2000). Pollen grains adhere to the papillar
cell (an epidermal derivative) and this interaction triggers the
further development of the male gametophyte (Lolle et al.,
1998). Pruitt et al. (2000) described the isolation and characterization of a gene, termed FIDDLEHEAD, whose product
normally functions in blocking both types of epidermal cell
interactions during vegetative development. The gene
encodes a protein that is predicted to synthesize long-chain
fatty acids. An interesting phenomenon is the promiscuous germination and growth of wild-type pollen on vegetative and
non-reproductive floral organs of a fiddlehead mutant (Lolle
and Cheung, 1993). These studies indicate that plants normally
restrict water flow across the cuticle in all tissues except the
stigma, and that the stigma cuticle may be unique in that it
can change its water permeability in response to interaction
with pollen.
The pollen coat
Disrupting pollen coat lipids or proteins in Brassicaceae
species can delay or block pollen hydration. In particular,
mutations that impair long-chain lipid synthesis, and consequently proper assembly of the pollen coating, severely
reduce the hydration of Arabidopsis pollen, resulting in male
sterility (Preuss et al., 1993; Hülskamp et al., 1995).
Hydraulic contact can be restored in these mutant grains by
the addition of purified triacylglycerides (Wolters-Arts et al.,
1998). Mutations that affect pollen coat proteins are less
extreme, perhaps because of partial functional redundancy.
1208
Firon et al. — Pollen water status, development and functioning
The lipid-rich stigma exudate of plants with wet stigmas is
thought to be at least partially functionally analogous to the
pollen coat (Dickinson, 1995). Mutations that eliminate this
stigma-secreted matrix cause female sterility, a defect that
can be bypassed by adding exogenous lipids (Goldman
et al., 1994; Wolters-Arts et al., 1998).
Mutants in lipid biosynthesis indicate a role for lipids in mediating water transfer at the stigma. For instance, the arabidopsis eceriferum (cer) mutants fail to hydrate on the stigma
because of decreased lipid content in their pollen coat – a
defect that can be overcome by high humidity or the addition
of triacylglycerides to the stigma (Preuss et al., 1993;
Wolters-Arts et al., 1998; Fiebig et al., 2000).
The arabidopsis pollen coat plays a vital role in mediating
the early contact between pollen grain and stigma. It protects
pollen grains from excess desiccation after dehiscence, contributes to pollen adhesion to the stigma and, most importantly,
facilitates hydration (Edlund et al., 2004). The pollen coat
oleosin domain protein GRP17 is required for rapid initiation
of hydration on the stigma (Mayfield and Preuss, 2000).
Other proteins present in the arabidopsis pollen coat include
extracellular lipases, two putative receptor kinases and a
potential Ca2+-binding protein (Mayfield et al., 2001).
Updegraff et al. (2009) have recently demonstrated that the
pollen coat protein extracellular lipase 4 is required for
efficient pollen hydration.
M O L E C U L A R M E C H A N I S M S I N VO LVE D
I N EN ABL IN G CH AN GE S I N POLL E N
WATER STAT US
In the following we describe mechanisms that have been
shown to exist in pollen which enable changes in pollen hydration level and protecting cellular components from damage
that includes protein denaturation and/or the harmful effects
of reactive oxygen species. It should be noted, however, that
the use/functionality of these mechanisms for maintaining
water homeostasis during each of the different phases of
pollen development is not yet established and that, in many
cases, their existence in pollen is based upon expression
analyses of the respective genes.
Water movement and water channels
Water can cross bilayers due to the lateral dynamics of phospholipids, and large-volume water translocation is facilitated
by water channels. Members of this family of membrane
channel proteins are called aquaporins and are present in
both the plasmalemma and the tonoplast membrane (Losch,
1999). Plant aquaporins are classified into four main
sub-families: plasma membrane-intrinsic proteins (PIPs),
tonoplast-intrinsic proteins (TIPs), nodulin26-like-intrinsic
proteins (NIPs) and small and basic membrane-intrinsic proteins (SIPs) (summarized by Forrest and Bhave, 2007; Soto
et al., 2008). Many of the TIPs and PIPs are water channels,
although some of the isoforms also transport solutes such as
glycerol, urea or boric acid (Forrest and Bhave, 2007; Soto
et al., 2008). Soto et al. (2008), hypothesizing that aquaporins
might play a significant and important role during pollen rehydration on the stigma, functionally characterized aquaporin
genes that are highly and selectively expressed in mature
pollen of Arabidopsis thaliana (within the top 10 % of
mature pollen-expressed genes). They found two TIP loci,
AtTip1;3 and AtTip5;1, confirming data from Bock et al.
(2006) who conducted a genome-wide analysis of transporter
genes expressed in the male gametophyte at four developmental stages. Using Xenopus oocyte-swelling assays, Soto et al.
(2008) established that AtTip1;3 and AtTip5;1 are bifunctional
aquaporins with intermediate levels of permeability to water
and high permeability to urea. To investigate a putative physiological role for aquaporins in water uptake and pollen tube
growth, four well-characterized A. thaliana PIP genes were
transiently expressed in lily pollen by particle bombardment
(Sommer et al., 2008). Expression of A. thaliana PIPs in lily
pollen showed that members of the PIP1 sub-family do not
confer enhanced water channel activity, whereas members of
the PIP2 sub-family do, suggesting that they may play a role
in pollen water transport. Aquaporins of the PIP2 class were
also suggested to be required for efficient anther dehydration
prior to dehiscence in tobacco (Bots et al., 2005).
Osmoregulation under water stress
Osmoregulation can lower water and osmotic potential by
several bars. Solutes accumulated during osmoregulation
consist of various carbohydrates, the compatible solute
proline and quaternary ammonium compounds.
Proline. The beneficial effect of an increased proline level lies
not only in its function as a solute and as a protective molecule
for dehydration-sensitive macromolecular surfaces, but also
in its ability to reduce the generation of free radicals
(Losch, 1999).
Sucrose. Pollen of some species, e.g. the palms Chamaerops
humilis and Trachycarpus fortunei, has a high sucrose
content (Speranza et al., 1997; Guarnieri et al., 2006) and survives for many days at high or low RH (Bassani et al., 1994;
Pacini et al., 1997). Sucrose also allows pollen to be stored at
low temperatures because it prevents the formation of ice crystals; pollen with low sucrose content, such as that from grasses
(Speranza et al., 1997), is difficult to store (Barnabas and
Rajki, 1981). Other possible functions for sucrose include
membrane protection against desiccation (Hoekstra et al.,
2001) and providing carbon skeletons during germination for
pollen tube wall formation. A system of this type is static
because it maintains the turgor pressure constant irrespective
of changes in humidity. Nevertheless, the system is probably
more complex because sucrose can be hydrolysed to glucose
and fructose, doubling turgor pressure. Indeed, variations in
the percentages of these sugars have been observed in
natural habitats and in the laboratory (Nepi et al., 2001;
Vesprini et al., 2002). This enables turgor pressure adjustments
in response to variations in temperature and RH during pollen
presentation and dispersal. Cytoplasmic polysaccharides and
even starch seem to be involved in this process of keeping
pollen viability high, at least in Helleborus (Vesprini et al.,
2002). The interconversion of carbohydrates seems to be
reduced or even absent in species with recalcitrant pollen
such as C. pepo and grasses, which have a generally low
sucrose content (Speranza et al., 1997). Such pollen soon
Firon et al. — Pollen water status, development and functioning
Concentration (mg g–1 d. wt)
160
140
120
100
80
60
40
20
0
A-9
A-7
A-5
A-3
A-1
A
Flower bud developmental stage
F I G . 4. Changes in sucrose concentration in developing pollen grains of tomato
‘Hazera 3017’. Sucrose concentration was analysed according to Hubbard et al.
(1990) as described in Firon et al. (2006), at six stages of development, 9, 7, 5, 3, 1
and 0 d before anthesis (A-9 to A-1 and A, respectively). Data represent the
average of at least three biological replicates. Vertical bars represent the s.e.
becomes non-viable, especially if humidity is low (Pacini
et al., 1997). Sucrose has been found to accumulate during
tomato pollen maturation and dehydration (Fig. 4), suggesting
its involvement in desiccation tolerance during pollen
water loss.
Interestingly, we found that tomato genotypes that exhibit
higher sucrose content in mature pollen grains exhibit higher
tolerance to heat stress (Firon et al., 2006). In all tested heatsensitive genotypes, the heat stress conditions (day/night temperatures of 31/25 8C) caused a marked reduction in starch
concentration in the developing pollen grains 3 d before anthesis, and a parallel decrease in the total sucrose concentration in
the mature pollen, whereas in the tested heat-tolerant
genotypes, starch accumulation 3 d before anthesis and
sucrose concentration at anthesis were not affected by heat
stress (Firon et al., 2006).
Trehalose. The potential role of trehalose as a protective sugar
against dehydration (Potts, 2001) should be mentioned,
though, to the best of our knowledge, there are no data available regarding its biosynthesis and accumulation during pollen
development. Research in this direction has a good chance of
contributing to our knowledge of pollen dehydration protective
mechanisms.
H+-ATPase activity. It was recently suggested that pollen grain
osmoregulation occurs via modulation of plasma membrane
H+-ATPase activity by 14-3-3 proteins (Pertl et al., 2010).
Increases in both plasma membrane-associated 14-3-3 proteins
and plasma membrane H+-ATPase activity were detected in
pollen grains challenged by hyperosmolar medium. In Lilium
pollen, H+-ATPase activity was shown to be important
during the stage at which the dehydrated pollen grains take
up water and have to adjust their turgor pressure to the water
potential of the surrounding stigma surface (Pertl et al., 2010).
Transporters
Solute import across the pollen plasma membrane, which
occurs via proteinaceous transporters, is required to support
1209
pollen development as well as for subsequent germination
and pollen tube growth. Results summarized by Bock et al.
(2006) stress the potential importance of the sucrose transporter SUC1 (At1g71880), a plasma membrane-localized H+/Suc
symporter widely expressed in vegetative organs. However,
transcriptome results show that it is the only member of the
SUC family that is highly expressed at the tricellular pollen
stage, during the last phase of pollen tube growth. The
plasma membrane proton pump AHA3 ( plasma membrane
H+-ATPase family), known to reside in phloem companion
cells (DeWitt and Sussman, 1995), was found to be selectively
expressed in microspores. Only half of the microspores from
heterozygous aha3 mutants developed into mature pollen
grains. This example suggests a distinct role for the plasma
membrane AHA3 in generating a proton gradient to drive
uptake of sugars and other nutrients (Robertson et al., 2004)
to nourish developing microspores. Schwacke et al. (1999) isolated putative transporters for proline; the uptake and accumulation of proline in developing and germinating pollen in
tomato plants correlated with the induction of a pollen-specific
member of the proline transporter ProT family, LeProT1.
Aside from its presumed role in osmotic adjustment and as a
compatible solute that protects proteins, proline may function
as a sink for energy and reducing equivalents, as a source of
nitrogen upon relief of stress, as a means of reducing acidity
and as a radical scavenger (Schwacke et al., 1999, and references therein). Biochemical characterization revealed that in
addition to proline, LeProT1 is an efficient transporter of
glycine betaine and the stress-induced amino acid
g-aminobutyric acid.
LEA proteins and dehydrins
In orthodox seeds, quiescence is acquired during the later
stages of maturation, when about 90 % of the water has been
lost by desiccation (Finkelstein et al., 2002). Acquisition of
desiccation tolerance is marked by a variety of factors, such
as an increase in low molecular weight solutes and the appearance of dehydrins; the D11 family of LEA proteins (Ismail
et al., 2010, and references therein). Dehydrins are very rich
in glycine residues and they are characterized by highly conserved 15-mer lysine-rich sequences termed K segments.
The K segment can form a putative amphipathic a-helical
structure, with the potential for both hydrophilic and hydrophobic interactions (Abba et al., 2006, and references
therein). Due to this property, dehydrins have a potential
chaperone-like function in stabilizing partially denatured proteins or membranes, coating them with a cohesive water
layer and preventing their coagulation during desiccation.
Indeed, Wolkers et al. (2001) suggested that LEA proteins
play a role together with carbohydrates in the formation of a
tight hydrogen-bonded network in dehydrated pollen and, possibly, in other anhydrobiotic organisms. Aside from their suggested ion-binding property, LEA proteins would thus have a
structural role in that these proteins might serve as anchors
in a tight molecular network to provide stability to macromolecular and cellular structures in the cytoplasm of anhydrobiotes in the dry state. In a proteomic study of pollen, there
was considerable overlap in some of the major proteins
present in mature pollen grains and seeds (Grobei et al., 2009).
1210
Firon et al. — Pollen water status, development and functioning
Notably, both proteomes contained high levels of LEA proteins and chaperones (Grobei et al., 2009), consistent with
an expected role for these proteins in conferring relatively
stress-tolerant dormant states in both mature pollen grains
and seeds (Zinn et al., 2010).
In the cold acclimation process, a large number of Cor
(cold-responsive)/Lea genes are transcriptionally activated,
and the accumulated proteins and metabolites lead to protection of cell structure integrity and function from freezing
damage (Thomashow, 1999). Cor/Lea genes can be regulated
by the dehydration-responsive element-binding (DREB) proteins that make up a sub-family of the ethylene-responsive
element-binding factor family. The latter contains two main
sub-classes, DREB1 and DREB2, involved in two separate
signal transduction pathways under low temperature and dehydration, respectively (Agarwal et al., 2006). It is interesting to
note that in maturing tomato microspores, several DREB
family members (DREB1, DREB2 and DREB3) were found
to be expressed, with DREB1 exhibiting heat stress-regulated
expression (Frank et al., 2009).
Heat stress proteins (HSPs)
HSPs are assumed to help other proteins maintain or regain
their native conformation by stabilizing their partially
unfolded states (Kotak et al., 2007). Similar to other organisms, in plants, HSPs are expressed not only in response to
stress, but also during various developmental programmes,
including pollen maturation, zygotic embryogenesis and seed
maturation (zur Nieden et al., 1995; Waters et al., 1996;
Wehmeyer and Vierling, 2000).
In seeds, a sub-set of Hsp genes is expressed during seed development (Wehmeyer et al., 1996; Wehmeyer and Vierling,
2000; Hong and Vierling, 2001). These developmentally regulated Hsp genes accumulate late in the maturation phase. The
expression of particular isoforms of Hsp genes during seed development suggests that these might have a distinct function
during seed maturation and that they are regulated by a
defined developmental programme. Arabidopsis plants with a
desiccation-intolerant mutant allele of ABI3 (abi3-6;
Nambara et al., 1994) were shown to have no detectable
Hsp17.4-CI in mature dry green seeds (Wehmeyer and
Vierling, 2000). The absence of small HSPs correlates with
a desiccation-intolerant phenotype, suggesting that small
HSPs might be required for desiccation tolerance in arabidopsis seeds.
Available pollen proteome and transcriptome data (Noir
et al., 2005; Volkov et al., 2005; Frank et al., 2009) revealed
the expression of several HSPs, including HSP70 and several
chaperonins. Expression of HSPs is regulated by heat stress
transcription factors (HSFs; Kotak et al., 2007). Indeed,
several members of the HSF family were detected in developing tomato microspores/pollen, exhibiting low expression
levels under ambient temperature conditions (Frank et al.,
2009). A comprehensive analysis of the arabidopsis male gametophyte transcriptome revealed Hsf gene expression, although
this family of transcription factors was found to be underrepresented in the gametophyte (Honys and Twell, 2003,
2004).
Antioxidants
Studies of Noir et al. (2005) and Holmes-Davis et al.
(2005), producing the mature pollen proteome of arabidopsis,
indicated the accumulation of several proteins that are involved
in the scavenging of reactive oxygen species, including ascorbate peroxidase, several superoxide dismutase proteins, catalase, glutathione peroxidase, glutathione S-transferase and
thioredoxin. The presence of these proteins in mature pollen
grains may both serve the maturing microspores during the dehydration phase and protect the mature pollen during the subsequent phases of presentation, dispersal and rehydration. High
constitutive expression levels of five ascorbate peroxidase
genes were recently detected in maturing tomato microspores:
two chloroplastic (thylakoid) members, a peroxisomal member
and two cytosolic members (Frank et al., 2009), indicating
their importance during post-meiotic stages of pollen development, including the dehydration phase.
De Gara et al. (1993) demonstrated long ago that
Dasypyrum villosum (a wild species of Triticinae) pollen contains the ascorbic acid synthesis pathway and actively synthesizes this compound. High activity of ascorbate peroxidase and
monodehydroascorbate reductase was detected in the mature
pollen grains, 10-fold higher than the activity of catalase, suggesting that ascorbate peroxidase may be the main enzyme
removing hydrogen peroxide produced during cellular metabolism (De Gara et al., 1993). Sheoran et al. (2009) identified a
number of proteins associated with stress responses in mature
canola pollen, including catalase, peroxidase, GRP and LEA
proteins. Two isoforms of catalase, peroxidase and GRP
were found to be upregulated and two isoforms of catalase
and LEA proteins were found to be downregulated in germinating pollen. The authors suggested that catalases and peroxidases may have dual roles, in stress tolerance and calcium
signalling, during canola pollen germination and tube growth.
CON CLU DI NG REMA RK S
During their developmental programme and functioning,
pollen grains undergo remarkable changes in their hydration
state – experiencing an increase in volume and water
content while being immersed in the locular fluid, followed
by a dehydration phase during which a large proportion of
the cellular water is removed. Mature, dehydrated pollen can
survive in a dry state for extended periods of time and accommodate changes in the RH of the environment to which they
are exposed. Landing on a compatible stigma, pollen water
content will increase, in accordance with the level of the
mature pollen’s water content capacity and the ability of the
stigma to release water. These drastic changes in water
content require structural, physiological and molecular
mechanisms that enable adjustments in water level and accommodation of the accompanying changes in volume, membrane
and protein environment; such mechanisms must also enable
coping with side effects and reactions, such as the production
of reactive oxygen species, which may harm the cellular components. It is suggested that pollen grains will use these
mechanisms, which have a developmental role, for coping
with environmental stress conditions. Additional data are necessary, however, to validate the function of these mechanisms
Firon et al. — Pollen water status, development and functioning
during the various phases of pollen development and under
stress conditions. The potential existence of a system that controls pollen water homeostasis is suggested in this review. Data
are limited, however, and further research is necessary to identify the components that constitute such a system, namely the
‘water-status sensor’, the ‘messenger(s)’ that deliver the information and the ‘control point’ that processes this information.
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