**6. Pollen metabolism and development: role of vacuoles and plastids**

#### **6.1. Role of vacuoles**

Vacuoles appearing at several stages of pollen development are correlated with metabolic activity. Pollen mother cells, like undifferentiated meristematic cells, are originally devoid of vacuoles but at telophase II small roundish vacuoles start to develop. Vacuolisation can occur once or twice (depending on species) during further stages of development [18]. Cyclic vacuolisation is always followed by storage of starch in amyloplasts (Figure 3 B–D), which then leads to disappearance of vacuoles and formation of new cytoplasm. Vacuolisation plays a role in increasing the volume of the pollen grain with the formation of new cytoplasmic components such as mitochondria, amyloplasts, other cell components and cytoplasmic reserves. Vacuolisation therefore reflects metabolic activity in the developing microspores. Vacuolisation is also associated with the storage of pectins during intine cell wall synthesis [146]. In Arabidopsis a large vacuole is formed by fusion of smaller vacuoles; this large vacuole is converted to smaller vacuoles again after the first mitotic division [176]. Lytic vacuoles (lysosomes) are formed to degrade mitochondria, ribosomes and plastids [18]. Mature pollen has only small vesicles filled with carbohydrates, but in species producing pollinia rather than single pollen (e.g. massulate orchids) small vacuoles with watery content are present. Reduced vacuolisation at maturity may be required to reduce pollen size during presentation and dispersal [33]. Pollen vacuolisation is also affected by abiotic stresses such as drought and temperature stresses (Table 3). Heat stress was shown to reduce pollen release from anthers [177]. Vacuoles also store metabolites such as sugars and play a role in regulating sugar homeostasis, metabolic activity and growth processes [178]. Sucrose cleavage into hexoses by vacuolar invertases can regulate osmotic potential of cells [179] and this can be used as a defence mechanism against stresses such as drought (Table 3). Abiotic stresses in Arabidopsis induce vacuolar invertase, as well as a tonoplast-associated monosaccharide transporter (*ESL1*) in vascular parenchyma cells [180]. Regulation of cellular sugar fluxes between cytoplasm and vacuoles is important to regulate osmotic potential and pollen hydration and this could play a role under environmental stress conditions. Vacuolar invertases that are expressed in pollen grains have been identified [108, 109], but their role in regulating pollen metabolism under stress conditions requires further investigation.

#### **6.2. Role of plastids**

Plastids are commonly present as undifferentiated pro-plastids at the end of meiosis. They divide later to differentiate and accumulate starch [181, 182]. Plastid division occurs in the vegetative cell of pollen before starch engorgement. Usually, there are one or two waves of starch accumulation in amyloplasts during pollen grain development in gymnosperms and angiosperms [162, 181]. In some plant species, pro-plastids in the generative cells are degraded by lysosomes immediately after the first haploid mitosis [183]. Plastids also store fatty acids and alcohol intermediates for pollen wall synthesis, as evidenced by the male sterile mutant *defective pollen wall* (*dpw*) [184]. Starch stored in the amyloplasts of the vegetative cell is in most plants hydrolysed before anther opening and pollen dispersal (Figure 3C). Physico-chemical properties of starch in plants with two cycles of starch synthesis vary between and within species [185]. Mature pollen can be starchy or starchless, depending on the presence or absence of starch grains in the vegetative cell amyloplasts (Table 1). This can be characteristic for plant families [185]. In some plants that flower throughout the year in the same environment pollen grains are always starchless (e.g., *Mercurialis annua*) [186]. Vice versa, in the case of *Parietaria judaica* which flowers from springtime to autumn, the proportion of starchy and starchless grains varies according to the season [187].

#### **6.3. Adjustment of osmotic pressure and water balance in pollen**

**6. Pollen metabolism and development: role of vacuoles and plastids**

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

metabolism under stress conditions requires further investigation.

Plastids are commonly present as undifferentiated pro-plastids at the end of meiosis. They divide later to differentiate and accumulate starch [181, 182]. Plastid division occurs in the vegetative cell of pollen before starch engorgement. Usually, there are one or two waves of starch accumulation in amyloplasts during pollen grain development in gymnosperms and angiosperms [162, 181]. In some plant species, pro-plastids in the generative cells are degraded by lysosomes immediately after the first haploid mitosis [183]. Plastids also store fatty acids and alcohol intermediates for pollen wall synthesis, as evidenced by the male sterile mutant *defective pollen wall* (*dpw*) [184]. Starch stored in the amyloplasts of the vegetative cell is in most plants hydrolysed before anther opening and pollen dispersal (Figure 3C). Physico-chemical

Vacuoles appearing at several stages of pollen development are correlated with metabolic activity. Pollen mother cells, like undifferentiated meristematic cells, are originally devoid of vacuoles but at telophase II small roundish vacuoles start to develop. Vacuolisation can occur once or twice (depending on species) during further stages of development [18]. Cyclic vacuolisation is always followed by storage of starch in amyloplasts (Figure 3 B–D), which then leads to disappearance of vacuoles and formation of new cytoplasm. Vacuolisation plays a role in increasing the volume of the pollen grain with the formation of new cytoplasmic components such as mitochondria, amyloplasts, other cell components and cytoplasmic reserves. Vacuolisation therefore reflects metabolic activity in the developing microspores. Vacuolisation is also associated with the storage of pectins during intine cell wall synthesis [146]. In Arabidopsis a large vacuole is formed by fusion of smaller vacuoles; this large vacuole is converted to smaller vacuoles again after the first mitotic division [176]. Lytic vacuoles (lysosomes) are formed to degrade mitochondria, ribosomes and plastids [18]. Mature pollen has only small vesicles filled with carbohydrates, but in species producing pollinia rather than single pollen (e.g. massulate orchids) small vacuoles with watery content are present. Reduced vacuolisation at maturity may be required to reduce pollen size during presentation and dispersal [33]. Pollen vacuolisation is also affected by abiotic stresses such as drought and temperature stresses (Table 3). Heat stress was shown to reduce pollen release from anthers [177]. Vacuoles also store metabolites such as sugars and play a role in regulating sugar homeostasis, metabolic activity and growth processes [178]. Sucrose cleavage into hexoses by vacuolar invertases can regulate osmotic potential of cells [179] and this can be used as a defence mechanism against stresses such as drought (Table 3). Abiotic stresses in Arabidopsis induce vacuolar invertase, as well as a tonoplast-associated monosaccharide transporter (*ESL1*) in vascular parenchyma cells [180]. Regulation of cellular sugar fluxes between cytoplasm and vacuoles is important to regulate osmotic potential and pollen hydration and this could play a role under environmental stress conditions. Vacuolar invertases that are expressed in pollen grains have been identified [108, 109], but their role in regulating pollen

**6.1. Role of vacuoles**

**6.2. Role of plastids**

Like soluble sugars, starch stored in plastids can play a role in adjusting osmotic pressure, particularly during presentation and dispersal (Table 3). Stored carbohydrates in plastids or in the cytoplasm, soluble or insoluble, can be used to adjust turgor pressure and protect grains against desiccation [188]. Many genes are involved in starch biosynthesis throughout pollen development [189]. Drought and temperature stresses can severely affect starch accumulation, and absence of starch in mature pollen can be an indicator of pollen sterility [109, 110, 115, 190– 192]. Endogenous starch is consumed during the first phases of pollen tube emission when pollen tube growth is at the expense of pollen reserves [193, 194]. After this autotrophous phase, pollen grains obtain carbohydrates and other substances from the stigma and style. Starch presence is not a direct indication of carbohydrate reserves present in pollen; hydrolysis of starch from amyloplasts increases soluble sugar levels in the cytoplasm and sugars are stored in the vacuole [188, 193]. Carbohydrates derived from starch hydrolysis in starch-less pollen grains alleviates the effect of heat and humidity stress during presentation and dispersal [193]. Starch in plants is normally phosphorylated. A tomato mutant lacking starch phosphorylation activity (*Legwd*) fails to degrade starch for pollen germination, resulting in sterile pollen [182]. Hydrolysis of starch supplies soluble osmotically active sugars which, together with amino acids such as proline, provide osmotic adjustment [195] (Tables 3 and 4). Regulation of turgor pressure is an essential aspect of pollen tube growth and elongation [196]. Osmo-regulation during the late maturation phase may function in the dehydration of pollen. Pollen dehydra‐ tion is associated with the induction of proteins that play a role in drought response: dehydrins, aquaporins, heat shock and LEA proteins [197]. High levels of osmotin expression in mature tobacco pollen is another indicator of osmotic stress response [198]. Potassium ions [199, 200] and phospholipids can also regulate osmotic pressure and cell swelling in pollen [201]. Regulation of pollen osmotic potential and water content and the role carbohydrates play in this process are clearly important in pollen development. Abiotic stresses (cold, heat and drought) during meiosis affect sink strength of the tapetum [109, 110, 126, 191, 202], but the dynamics of carbohydrate metabolism at the gametophyte level remain poorly understood.



**Table 4.** Synthesis of osmotically active components in pollen, and their effect at different stages of development. Orthodox and recalcitrant grains could have a similar physiological behaviour until the onset of desiccation, but the amount and quality of the osmotic molecules and the activity of their biosynthetic enzymes distinguish the two categories in the later stages of pollen development.
