**2. Male gametophyte development and its biodiversity**

In angiosperms, male gametophytes develop in the anther. Each anther consists of two thecae, each consisting of two adjacent microsporangia that are separated by the connective tissue (Figure 1). The first phase in pollen development, the meiotic division of the sporophytic meiocytes of the four microsporangia to form haploid tetrads and young microspores, is called microsporogenesis (Figure 2). During the second phase, microgametogenesis, the microspores enlarge and become vacuolated. Vacuolisation and the cytoskeleton force the nucleus to migrate to a peripheral position. The first mitotic division is asymmetric and produces a germ cell that is engulfed by the cytoplasm of the vegetative cell to become physically isolated from the vegetative cell (bi-cellular pollen; cell-within-a-cell). The germ cell then undergoes a second mitotic division to produce the two sperm cells (Figure 2). During fertilisation, one male gamete fuses with the egg cell and the other with the two polar nuclei of the central cell to form the zygote and endosperm, respectively. The male sperm cells are very diminutive in size, but transcriptome analysis has recently revealed that their gene expression pattern is unlike any other plant tissue, suggesting that they are functionally very specialised [29].


**Table 1.** Classification of pollen diversity according to cytological events during pollen development, and examples of some representative plant species. Pollenkitt (PK) is typically present in zoophilous and entomophilous species and is generally absent in anemophilous species, with the exception of *Olea europaea*, a secondary anemophilous species. Some plant families (e.g. Liliaceae) have a majority of members with two-celled starchless grains and some species with three-celled starchless pollen grains [30, 185] (E. Pacini, personal observations).

The Trials and Tribulations of the Plant Male Gametophyte — Understanding Reproductive Stage Stress Tolerance http://dx.doi.org/10.5772/61671 707

**2. Male gametophyte development and its biodiversity**

706 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

other plant tissue, suggesting that they are functionally very specialised [29].

Starchy <sup>⋅</sup>*Olea europaea* (Oleaceae) PK ⋅ *Erica arborea* (Ericaceae)

> pollination syndrome) ⋅ Lamiaceae (PK) ⋅ Myrtaceae (PK) ⋅ Scrophulariaceae (PK)

> ⋅ *Plantago* sp. (PK) ⋅ *Portulaca tuberosa* (PK)

Starchless <sup>⋅</sup>*Laurus nobilis* PK ⋅ Malvaceae PK

with three-celled starchless pollen grains [30, 185] (E. Pacini, personal observations).

⋅ Solanaceae (PK presence depends on

⋅ *Acanthus mollis* (Acanthaceae) (PK) ⋅ *Bryonia dioica* (Cucurbitaceae) (PK) ⋅ *Cucumis melo* (Cucurbitaceae) (PK) ⋅ Liliaceae (some species) (PK)

⋅ *Cucurbita pepo* (Cucurbitaceae) (PK)

**Table 1.** Classification of pollen diversity according to cytological events during pollen development, and examples of some representative plant species. Pollenkitt (PK) is typically present in zoophilous and entomophilous species and is generally absent in anemophilous species, with the exception of *Olea europaea*, a secondary anemophilous species. Some plant families (e.g. Liliaceae) have a majority of members with two-celled starchless grains and some species

*Pollen type*

**Orthodox (>20% water)**

**Recalcitrant (<20% water)**

**Starch content**

Starchless

Starchy

In angiosperms, male gametophytes develop in the anther. Each anther consists of two thecae, each consisting of two adjacent microsporangia that are separated by the connective tissue (Figure 1). The first phase in pollen development, the meiotic division of the sporophytic meiocytes of the four microsporangia to form haploid tetrads and young microspores, is called microsporogenesis (Figure 2). During the second phase, microgametogenesis, the microspores enlarge and become vacuolated. Vacuolisation and the cytoskeleton force the nucleus to migrate to a peripheral position. The first mitotic division is asymmetric and produces a germ cell that is engulfed by the cytoplasm of the vegetative cell to become physically isolated from the vegetative cell (bi-cellular pollen; cell-within-a-cell). The germ cell then undergoes a second mitotic division to produce the two sperm cells (Figure 2). During fertilisation, one male gamete fuses with the egg cell and the other with the two polar nuclei of the central cell to form the zygote and endosperm, respectively. The male sperm cells are very diminutive in size, but transcriptome analysis has recently revealed that their gene expression pattern is unlike any

*Two-celled Three-celled*

(PK)

(PK)

⋅ *Wolfia arrhiza* (Araceae) (PK) ⋅ *Lilium bienne* (Liliaceae) (PK) ⋅ *Nelumbo nucifera* (Nelumbonaceae)

⋅ *Hedera helix* (Araliaceae) (PK) ⋅ *Borago officinalis* (Boraginaceae)

⋅ *Canna indica* (Cannaceae) (PK) ⋅ *Tulipa gesneriana* (Liliaceae) (PK)

⋅ Caprifoliaceae (PK) ⋅ Asteraceae (PK)

⋅ Amaranthaceae (PK) ⋅ Alismataceae (PK)

⋅ *Cereus* sp. (Cactaceae) (PK) ⋅ Caryophillaceae (PK)

⋅ Poaceae

**Figure 2. Male gametophyte development in angiosperms.** Pollen grains develop in the stamen, which consists of a filament supporting the anther. The vascular bundles in the filament conduct nutrients from the mother plant to the anther. The cross-section of the anther (rice) before the onset of meiosis shows the four microsporangia where the male gametophytes develop. From outside to inside, the anther wall consists of the epidermis, the endothecium, the middle layer and the tapetum. Both the middle layer and the tapetum degenerate towards pollen maturity, leaving only the epidermis and the endothecium to protect the pollen grains in the loculus before anther opening. The central cells of the anther, the pollen mother cells (meiocytes), differentiate and become selectively isolated from the mother plant through callose secretion by the meiocyte cytoplasm. The pollen mother cells undergo meiosis to form tetrads. The uninucleate young microspores are released from the tetrad with the help of enzymes secreted by the tapetum [313]. Exine is completed with the intervention of polymers secreted by the tapetum in the loculus [35]. Young microspores have a central nucleus and in Poaceae they are with the pore attached to tapetum until anther opening. The germination pore becomes visible and a large vacuole forms, pushing, with the intervention of the cytoskeleton, the nucleus in a periph‐ eral position (polarised microspore stage) [176, 314]. At the vacuolated stage, the microspores undergo an asymmetric division (pollen mitosis I) to produce the vegetative and generative nucleus. The generative nucleus is then isolated in a separate compartment within the vegetative cell to form a bi-cellular pollen grain (cell-within-a-cell). During pollen maturation, the vacuole of the vegetative cell gradually decreases in size and accumulation of starch granules is ob‐ served (engorgement). In plants with tri-cellular pollen, a second mitotic division of the germ cell takes place before anthesis (pollen mitosis II) to produce the two sperm cells. At this stage, the two germ cells are found in close proximi‐ ty of the vegetative nucleus (male germ unit).

Nuclei number (meiosis, tetrad, microspores), pollen grain cell number (bi-cellular and tricellular pollen) and other cytological events (vacuolisation, starch accumulation/hydrolysis, water content) are used to determine pollen developmental stages (Figure 2). These parameters can differ between plant species and differences in pollen development can be used for systematic classifications (Table 1). At dispersal, angiosperm pollen grains can be bi-cellular or tri-cellular (Table 2) [30]. In tri-cellular pollen, the second mitotic division occurs prior to dispersal and pollen is dispersed with the two sperm cells already formed (Figure 2). In bicellular pollen, the second mitotic division occurs during pollen tube growth inside the stylestigma. The term male germ unit describes the relative position and cytological connections between the generative cell, the sperm cells and the vegetative cell nucleus in the mature pollen and pollen tube [2, 31]. Very few species release bi- and tri-cellular pollen grains at the same time. When this occurs (e.g. *Annona cherimola*), the ratio between bi-cellular and tri-cellular pollen grains was shown to depend on environmental factors such as temperature regime and relative humidity during the last phases of maturation [32]. Tri-cellular pollen grains have completed their development before dispersal and are typical for plant families that include important dicot and monocot crop species such as Asteraceae, Lamiaceae, Brassicaceae and Poaceae (Table 1). In some plants, pollen is dispersed as aggregates containing a high number of pollen grains (e.g. massulate orchids) [33]. Orchids are monocots that produce bicellular pollen; the generative cell is spherical at dispersal but changes to the normal spindle shape prior to the second mitotic division when pollen lands on the stigma and starts emitting the pollen tube [34]. Pollen development is further subdivided in early, middle and late stages according to cytological and morphological features such as the presence of a vacuole (Table 1; Figure 2) [35–37]. Vacuolisation occurs only once in some species, but twice in others (once during the early microspore to bi-cellular stage and once during early bicellular to late microspore stage) [18]. Stages of pollen vacuolisation alternate with stages of starch accumu‐ lation in plastids (engorgement) and starch accumulation can therefore also occur once or twice. Mature pollen grains can be starchy or starch-less depending on whether starch is present in mature exposed pollen grains (Table 1). Another classification is based on water content of pollen at dispersal: orthodox and recalcitrant pollen is dispersed in partially desiccated or partially hydrated form, respectively. Other differences concern the presence or absence of pollenkitt that distinguish animal/ insect from wind pollinators, respectively (with rare exceptions; Table 1). The diversity in pollen development between different plant species is complex and is functionally important. Different mechanisms have evolved under a variety of environmental constraints to secure pollination success and survival of the species.

#### **3. Meiosis: The start of reproductive development**

The decision to flower in higher plants is carefully controlled by environmental stimuli such as temperature and photoperiod [38–42]. After floral meristem initiation and formation of flower buds, meiosis is the committed step for sexual reproduction and formation of the gametophytes. The onset of meiosis is regulated by signals coming from the mother plant. Sugar availability plays an important role in driving cell division by inducing expression of The Trials and Tribulations of the Plant Male Gametophyte — Understanding Reproductive Stage Stress Tolerance http://dx.doi.org/10.5772/61671 709


Nuclei number (meiosis, tetrad, microspores), pollen grain cell number (bi-cellular and tricellular pollen) and other cytological events (vacuolisation, starch accumulation/hydrolysis, water content) are used to determine pollen developmental stages (Figure 2). These parameters can differ between plant species and differences in pollen development can be used for systematic classifications (Table 1). At dispersal, angiosperm pollen grains can be bi-cellular or tri-cellular (Table 2) [30]. In tri-cellular pollen, the second mitotic division occurs prior to dispersal and pollen is dispersed with the two sperm cells already formed (Figure 2). In bicellular pollen, the second mitotic division occurs during pollen tube growth inside the stylestigma. The term male germ unit describes the relative position and cytological connections between the generative cell, the sperm cells and the vegetative cell nucleus in the mature pollen and pollen tube [2, 31]. Very few species release bi- and tri-cellular pollen grains at the same time. When this occurs (e.g. *Annona cherimola*), the ratio between bi-cellular and tri-cellular pollen grains was shown to depend on environmental factors such as temperature regime and relative humidity during the last phases of maturation [32]. Tri-cellular pollen grains have completed their development before dispersal and are typical for plant families that include important dicot and monocot crop species such as Asteraceae, Lamiaceae, Brassicaceae and Poaceae (Table 1). In some plants, pollen is dispersed as aggregates containing a high number of pollen grains (e.g. massulate orchids) [33]. Orchids are monocots that produce bicellular pollen; the generative cell is spherical at dispersal but changes to the normal spindle shape prior to the second mitotic division when pollen lands on the stigma and starts emitting the pollen tube [34]. Pollen development is further subdivided in early, middle and late stages according to cytological and morphological features such as the presence of a vacuole (Table 1; Figure 2) [35–37]. Vacuolisation occurs only once in some species, but twice in others (once during the early microspore to bi-cellular stage and once during early bicellular to late microspore stage) [18]. Stages of pollen vacuolisation alternate with stages of starch accumu‐ lation in plastids (engorgement) and starch accumulation can therefore also occur once or twice. Mature pollen grains can be starchy or starch-less depending on whether starch is present in mature exposed pollen grains (Table 1). Another classification is based on water content of pollen at dispersal: orthodox and recalcitrant pollen is dispersed in partially desiccated or partially hydrated form, respectively. Other differences concern the presence or absence of pollenkitt that distinguish animal/ insect from wind pollinators, respectively (with rare exceptions; Table 1). The diversity in pollen development between different plant species is complex and is functionally important. Different mechanisms have evolved under a variety

708 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

of environmental constraints to secure pollination success and survival of the species.

The decision to flower in higher plants is carefully controlled by environmental stimuli such as temperature and photoperiod [38–42]. After floral meristem initiation and formation of flower buds, meiosis is the committed step for sexual reproduction and formation of the gametophytes. The onset of meiosis is regulated by signals coming from the mother plant. Sugar availability plays an important role in driving cell division by inducing expression of

**3. Meiosis: The start of reproductive development**

**Table 2.** Table showing the presence and abundance of locular space and fluid and relationship to pollen dispersal units in angiosperms. The locular fluid volume is extremely reduced when the pollen dispersal unit is of the compound type. Locular space and fluid are present from meiotic prophase until anther desiccation and opening.

the cell cycle regulatory protein cyclin that induces meiosis [43–46]. The initiation of meiosis to form the male and female gametophytes in the anther and ovary is normally a synchronised process [47–52]. However, this is not always the case in some plants and abiotic stresses can cause asynchrony between male and female meiosis [47, 51]. Most commonly, in aseptate anthers all sporogenous initials will proceed to undergo meiosis, while in septate anthers only some initials will undergo meiotic division [53]. This difference will affect locular space and liquid volume available to pollen, pollen number per locule and ultimately the dispersal unit (Table 2). After meiosis, male and female gametophytes follow a very different path of development. While ovule development and maturation is a gradual process, formation of large amounts of pollen grains in the anthers is energetically more demanding. At the time of meiosis, the anthers represent the highest sink strength in the flower and anthers are known to have the highest soluble sugar content of any plant tissue [54, 55]. Synchrony of male meiosis can also be affected in interspecific hybrids [50]. Pollen sterility caused by meiotic asynchrony is a major problem in interspecific rice hybrids where productivity is affected [56]. Mutagenesis approaches in model plants are gradually revealing genes that are involved in initiating meiosis and its progression through the different phases [57–61]. Silencing of the antherspecific zinc finger transcription factor *MEZ1* causes abnormal meiosis and pollen abortion in petunia [62]. The Arabidopsis *STUD*, *TAM, DUET, MALE MEIOCYTE DEATH1, AtKIN14a, b* and *TETRASPORE* genes are responsible for different aspects of male meiosis, such as maintaining pace, synchrony, chromosome organisation and transition between different stages [63–68].

Pollen biotechnology is a potentially powerful tool for crop breeding. Genes that regulate progression and synchrony of pollen meiosis and their regulation (e.g. effect of abiotic stresses) can be exploited for establishing hybrid breeding technologies, for instance, using mutant lines that are conditionally arrested at pollen meiosis [69, 70]. Progress in understanding pollen meiosis will be accelerated by more refined technologies that make it possible to study the meiotic transcriptome in detail [71]. Transcriptome profiling has been used to investigate the

**Figure 4.** Change of shape and size of pollen according to their water content during the more critical phases of dehy‐ dration, presentation, dispersal and rehydration. High temperature and low relative humidity affect desiccation-sensi‐ tive pollen (DS) more than desiccation-resistant pollen (DR).

effect of abiotic stresses on pollen meiosis and pollen development [72, 73]. Abiotic stresses such as cold during meiosis can lead to formation of diploid gametes [74]. Polyploidisation and manipulation of chromosome number during meiosis can be used to increase diversity in breeding of crop plants [75, 76]. Some Arabidopsis mutants (*DIF1*, *TETRASPORE*, *PARALLEL SPINDLE1* and *Jason*) that affect ploidy levels can improve our understanding of pollen meiosis and how it is affected by the environment [66, 77–80].

#### **4. The importance of the anther tapetum**

The tapetum surrounds the pollen mother cells before meiosis and is the inner cell layer of the anther wall (Figure 2). The tapetum plays an important role in pollen develop‐ ment: it secretes the locular fluid containing nutrients for pollen development and depos‐ its components of the pollen cell wall. When these functions are fulfilled, the tapetum undergoes a natural programmed cell death response (PCD) [81–83]. This process is essential to sustain pollen development: PCD generates nutrients for the locular fluid to feed the native pollen grains [81, 83–88]. Tapetum cells are generally polyploid and/or multinucleate and are metabolically very active. Tapetal-specific gene transcripts are the most prevalent fraction of total anther transcripts [89]. Polyploidisation and genome endoduplication are commonly observed in plant tissues with high metabolic activity [90]. High


effect of abiotic stresses on pollen meiosis and pollen development [72, 73]. Abiotic stresses such as cold during meiosis can lead to formation of diploid gametes [74]. Polyploidisation and manipulation of chromosome number during meiosis can be used to increase diversity in breeding of crop plants [75, 76]. Some Arabidopsis mutants (*DIF1*, *TETRASPORE*, *PARALLEL SPINDLE1* and *Jason*) that affect ploidy levels can improve our understanding of pollen meiosis

**Figure 4.** Change of shape and size of pollen according to their water content during the more critical phases of dehy‐ dration, presentation, dispersal and rehydration. High temperature and low relative humidity affect desiccation-sensi‐

The tapetum surrounds the pollen mother cells before meiosis and is the inner cell layer of the anther wall (Figure 2). The tapetum plays an important role in pollen develop‐ ment: it secretes the locular fluid containing nutrients for pollen development and depos‐ its components of the pollen cell wall. When these functions are fulfilled, the tapetum undergoes a natural programmed cell death response (PCD) [81–83]. This process is essential to sustain pollen development: PCD generates nutrients for the locular fluid to feed the native pollen grains [81, 83–88]. Tapetum cells are generally polyploid and/or multinucleate and are metabolically very active. Tapetal-specific gene transcripts are the most prevalent fraction of total anther transcripts [89]. Polyploidisation and genome endoduplication are commonly observed in plant tissues with high metabolic activity [90]. High

and how it is affected by the environment [66, 77–80].

tive pollen (DS) more than desiccation-resistant pollen (DR).

710 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

**4. The importance of the anther tapetum**

**Table 6.** Table showing the main categories of monad pollen, their size, shape at dispersal, time for rehydration and germination of orthodox (oval) and recalcitrant (spherical) pollen grains, including representative examples and some ecological consequences. The average pollen diameter is 30–100 micrometers with low water content. Orthodox and recalcitrant grains have ecological devices to reduce water loss during presentation and dispersal, e.g. pollen presentation by anthers that are enclosed by the flower corolla and exposing anthers outside the flower as for poricidal anthers.

metabolic activity of tapetum cells is required during meiosis for production of callose, a temporary cell wall that separates the microspores from the tetrad, and for biosynthesis and secretion of sporopollenin for the exine pollen cell wall [91–93]. Mutations that affect callose deposition and dissolution affect microspore development and fertility [94, 95]. The main tapetum nutritional activity occurs during the microspore stage and the first signs of degeneration do not occur at the same stage in different species [96] but degeneration normally reaches completion near the end of the uni-nucleate microspore stage [97].

The secretory tapetum is the most common type [97]. The tapetum cells form the inner lining of the loculus and remain in place until they degenerate. In some plant species (e.g. Poaceae), the young microspores are found to attach themselves to the tapetum inner wall [35, 98]. In the secretory tapetum, the inner cell wall directed towards the loculus and the radial walls dissolve using a natural protoplasting event to facilitate the secretory function. Orbicules or Ubisch bodies are secreted towards the loculus by the tapetum cytoplasm; their function is not yet elucidated and only unproven hypotheses as to their role have been put forward [99–101]. During development, microspores are dispersed in the locular fluid, the volume of which can vary widely according to anther morphology (aseptate or septate) and the type of pollen dispersal units: more locular fluid is generally present in aseptate anthers and/or when pollen are dispersed as single units, while less fluid is present when pollen are dispersed as aggregates (Table 2) [82]. When released from the tetrad, pollen grains are in direct contact with the secretory tapetum [82]. The substances that are secreted in the locular fluid are neutral polysaccharides, pectins, proteins and lipids, and their relative proportion varies during pollen development [102]. The amount of locular fluid secreted depends also on the number, size and shape of the pollen grains and the dispersal unit (monads *vs*. polyads; Table 2).

Another form of tapetum is the amoeboid or periplasmodial tapetum which is, for example, found in the Asteraceae family [82]. In this case, the tapetum cell layer undergoes a reorgani‐ sation rather than degeneration during its early development. During meiosis, the tapetal cells form long extensions that engulf individual pollen mother cells. At the tetrad stage, the tapetum reorganises to form a periplasmodium which separates the individual young microspores and encloses them within a vacuole in the tapetal cytoplasm [103]. The amoeboid tapetum, better than the more common secretory type, illustrates the nurturing function of the tapetum.

The tapetum forms the interface between the sporophyte and the male gametophyte and is therefore in a strategic position to control reproductive development. Some of the substances entering the tapetum come from the external cell layers of the anther and other parts of the mother plant [104]. The mother plant supplies nutrients via the vascular bundle of the anther filament [84, 105]. Downloading occurs in the anther connective tissue cells and transport to the middle layer occurs symplastically [83]. The outer anther wall cells are connected via plasmodesmata, but the tapetum layer is symplastically isolated from other anther wall cells. Delivery of sugars into the tapetum requires apoplastic transport [55, 106, 107]. The apoplastic cell wall invertase gene is expressed in the tapetum and is responsible for mobilising sucrose into the tapetum cells [108, 109]. Repression of tapetal cell wall invertase activity and gene expression by different abiotic stresses blocks sugar transport to the pollen grains [108–112]. At least in some species nutritive substances are stored temporarily in the tapetum and are then absorbed by the developing pollen grains [102, 113].

The meiotic stage of pollen development is very sensitive to cold, heat and drought stress (Table 3) [25, 28, 109, 110, 114, 115]. It is likely that abiotic stresses at the time when the tapetum is metabolically most active interfere with the synthesis of pollen cell wall components and degeneration do not occur at the same stage in different species [96] but degeneration normally reaches completion near the end of the uni-nucleate microspore stage [97].

712 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

The secretory tapetum is the most common type [97]. The tapetum cells form the inner lining of the loculus and remain in place until they degenerate. In some plant species (e.g. Poaceae), the young microspores are found to attach themselves to the tapetum inner wall [35, 98]. In the secretory tapetum, the inner cell wall directed towards the loculus and the radial walls dissolve using a natural protoplasting event to facilitate the secretory function. Orbicules or Ubisch bodies are secreted towards the loculus by the tapetum cytoplasm; their function is not yet elucidated and only unproven hypotheses as to their role have been put forward [99–101]. During development, microspores are dispersed in the locular fluid, the volume of which can vary widely according to anther morphology (aseptate or septate) and the type of pollen dispersal units: more locular fluid is generally present in aseptate anthers and/or when pollen are dispersed as single units, while less fluid is present when pollen are dispersed as aggregates (Table 2) [82]. When released from the tetrad, pollen grains are in direct contact with the secretory tapetum [82]. The substances that are secreted in the locular fluid are neutral polysaccharides, pectins, proteins and lipids, and their relative proportion varies during pollen development [102]. The amount of locular fluid secreted depends also on the number, size and

shape of the pollen grains and the dispersal unit (monads *vs*. polyads; Table 2).

then absorbed by the developing pollen grains [102, 113].

tapetum.

Another form of tapetum is the amoeboid or periplasmodial tapetum which is, for example, found in the Asteraceae family [82]. In this case, the tapetum cell layer undergoes a reorgani‐ sation rather than degeneration during its early development. During meiosis, the tapetal cells form long extensions that engulf individual pollen mother cells. At the tetrad stage, the tapetum reorganises to form a periplasmodium which separates the individual young microspores and encloses them within a vacuole in the tapetal cytoplasm [103]. The amoeboid tapetum, better than the more common secretory type, illustrates the nurturing function of the

The tapetum forms the interface between the sporophyte and the male gametophyte and is therefore in a strategic position to control reproductive development. Some of the substances entering the tapetum come from the external cell layers of the anther and other parts of the mother plant [104]. The mother plant supplies nutrients via the vascular bundle of the anther filament [84, 105]. Downloading occurs in the anther connective tissue cells and transport to the middle layer occurs symplastically [83]. The outer anther wall cells are connected via plasmodesmata, but the tapetum layer is symplastically isolated from other anther wall cells. Delivery of sugars into the tapetum requires apoplastic transport [55, 106, 107]. The apoplastic cell wall invertase gene is expressed in the tapetum and is responsible for mobilising sucrose into the tapetum cells [108, 109]. Repression of tapetal cell wall invertase activity and gene expression by different abiotic stresses blocks sugar transport to the pollen grains [108–112]. At least in some species nutritive substances are stored temporarily in the tapetum and are

The meiotic stage of pollen development is very sensitive to cold, heat and drought stress (Table 3) [25, 28, 109, 110, 114, 115]. It is likely that abiotic stresses at the time when the tapetum is metabolically most active interfere with the synthesis of pollen cell wall components and the secretion of the locular fluid. This may cause abortion of the young microspores. The formation of the locular fluid is associated with an increase in pollen volume and increased vacuolisation, a process that is affected by water stress (Table 3) [28]. The presence of abundant locular fluid (e.g. Solanaceae and Poaceae) or its extreme reduction (e.g. some orchids, Fabaceae and Myrtaceae; Table 2) has so far not been correlated with higher or lower tolerance to drought stress. Plant species with a periplasmodial tapetum have a reduced volume of locular fluid. In this case, each microspore is engulfed in the tapetum cytoplasm, so pollen nutrition is direct and does not require an abundant locular fluid [82]. Abiotic stresses may interfere with tapetal PCD and affect its functionality [87]. Both premature and retarded degeneration of the tapetum cause pollen sterility [83, 87, 116–119]. Production of reactive oxygen species (ROS) has recently been implicated in the regulation of PCD timing in the tapetum [120]. ROS are produced in response to many abiotic stresses [121]. Premature tapetum degeneration is a major cause of pollen sterility and yield loss under abiotic stress conditions [118, 122–125]. Carbohydrate mobilisation to the tapetum and its genetic control may play an important role in guaranteeing pollen development under stress conditions. Anther sink strength is reduced in stress-sensitive species [108–110, 126]. At the same time, sugars appear to be redirected to other tissues, e.g. leading to starch accumulation in the endothecium layer of the anther wall [106, 107, 127]. The tapetum is a sporophytic tissue and its function is controlled by signals from the sporophyte (sugars, hormones). Improvement of stress tolerance in crop species will therefore require a better understanding of the effect of stress on the sporophyte, as well as on sporophyte–gametophyte communication.



**Table 3.** Effect of abiotic stresses on different stages of pollen development, and possible evolutionary defence mechanisms of the male gametophyte.
