**9. Pollen dehydration, presentation and anther dehiscence**

#### **9.1. Orthodox pollen and cross-pollination**

In cross-pollinating plants, the flower opens at anthesis and the pollen is dispersed to reach other plants (chasmogamy). To survive dispersal in the environment, pollen needs to be in a dehydrated state with low metabolic activity (Figure 4) [217, 218]. This is the case for orthodox pollen which is dehydration-tolerant and is dispersed with low water content (<20%). Ortho‐ dox pollen can travel over larger distances without losing viability [160, 219]. Near anthesis, rapid extension of the anther filament seals the xylem, interrupting sap flow to the anther. The phloem redistributes the locular content to other plant parts [160, 220, 221]. The epidermis and endothecium layers of the anther wall dehydrate and pollen grain hydration levels reach an equilibrium with the environment [222]. Environmental parameters such as temperature and relative air humidity influence pollen water content [186] and osmotic adjustment is used to balance water content in function of environmental conditions (Table 4) [27]. Orthodox pollen also has low metabolic homeostasis to prevent cellular damage during dispersal [160]. The duration of developmental arrest and viability of pollen depends on environmental conditions at dispersal and the type of reserve substances present in the pollen (Table 4) [27, 188]. These defence mechanisms protecting pollen grains during presentation, dispersal and pollination vary depending on the degree and duration of dehydration during dispersal and depend on whether plants are anemo- or zoophilous pollinators (Table 5) [27, 160, 223]. Relative air humidity can adversely affect pollination efficiency because absorption of water from the environment can lead to precocious pollen tube emission when the correct hydrated state is reached (Figure 3E) [160]. Entomophilous pollen is also affected by compounds that are secreted by the insect carrier (e.g. bees) [224–226]. Plants producing orthodox pollen are potentially out-crossing; both out-crossing and self-pollination can occur in these plant species, unless there is a self-incompatibility system in place to prevent self-pollination [227].


**Table 5.** Common types of modalities present in different angiosperms in order to reduce and/or avoid the harmful effects of the environment during pollen presentation and dispersal.

#### **9.2. Recalcitrant pollen and self-pollination**

can be paused once or twice at the microspore or bicellular stages. The ability of pollen development to be interrupted is an adaptation mechanism to protect pollen against extreme environmental conditions during summer or winter. Interruptions are more likely to occur in plants where pollen development takes longer, especially in temperate climates where unexpected harsh weather conditions can occur. Some gymnosperms (e.g. *Juniperus commu‐ nis*) and woody perennial angiosperms (e.g. birch, elder and hazelnut) that disperse their pollen at the end of winter differentiate their flower buds in autumn when environmental conditions are favourable [214, 215]. Under severe winter conditions, flower development is arrested and resumed in early spring. In hazelnuts, this interruption occurs at the bi-cellular stage [215]. The developing pollen grains appear dormant and anther metabolism is repressed. The influx of substances from the mother plant and the activity of anther wall chloroplasts are also reduced, suggesting that developmental arrest may be regulated by the mother plant. In some species, developmental arrest occurs prior to pollen meiosis. In some Mediterranean plants, flower buds develop during late spring but stay dormant during the hot and dry summer and development resumes in autumn [216]. The dioecious bay laurel (*Laurus nobilis*) flower buds of both sexes develop in early autumn, they pause development in winter and flower ripening and pollination occurs during early springtime [163]. It is not known how this developmental arrest of pollen development is controlled at the molecular and physiological level, but it provides a powerful defence mechanism to protect pollen and maintain fertility

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

under sub-optimal climatic conditions.

**9.1. Orthodox pollen and cross-pollination**

**9. Pollen dehydration, presentation and anther dehiscence**

In cross-pollinating plants, the flower opens at anthesis and the pollen is dispersed to reach other plants (chasmogamy). To survive dispersal in the environment, pollen needs to be in a dehydrated state with low metabolic activity (Figure 4) [217, 218]. This is the case for orthodox pollen which is dehydration-tolerant and is dispersed with low water content (<20%). Ortho‐ dox pollen can travel over larger distances without losing viability [160, 219]. Near anthesis, rapid extension of the anther filament seals the xylem, interrupting sap flow to the anther. The phloem redistributes the locular content to other plant parts [160, 220, 221]. The epidermis and endothecium layers of the anther wall dehydrate and pollen grain hydration levels reach an equilibrium with the environment [222]. Environmental parameters such as temperature and relative air humidity influence pollen water content [186] and osmotic adjustment is used to balance water content in function of environmental conditions (Table 4) [27]. Orthodox pollen also has low metabolic homeostasis to prevent cellular damage during dispersal [160]. The duration of developmental arrest and viability of pollen depends on environmental conditions at dispersal and the type of reserve substances present in the pollen (Table 4) [27, 188]. These defence mechanisms protecting pollen grains during presentation, dispersal and pollination vary depending on the degree and duration of dehydration during dispersal and depend on whether plants are anemo- or zoophilous pollinators (Table 5) [27, 160, 223]. Relative air humidity can adversely affect pollination efficiency because absorption of water from the

In self-pollinating plants, pollen does not have to travel far to pollinate and therefore does not need to undergo severe dehydration at maturity. These plants produce recalcitrant pollen grains which are dispersed with high relative water content (30–70%); pollen remains meta‐ bolically active at dispersal and continues to develop to the point of germination (reduced developmental arrest). Recalcitrant pollen grains are dehydration-sensitive and are typically very short-lived and highly sensitive to variation in relative air humidity [160] (Figure 3 A1 and A2; Figure 4). However, cross-pollination with recalcitrant pollen is possible but is restricted to proximate flowers only [228]. Some plant species produce both chasmogamic and cleistogamic flowers, thereby increasing the chance of reproductive success [227]. In crop species (e.g. wheat, barley, rice), cleistogamic breeding systems may have been selected during domestication to limit gene flow and preserve preferred gene combinations [229–233]. The absence of pollen presentation in cleistogamic self-pollinating plants is thought to be a protection against abiotic stresses such as drought and heat, as pollen number is considered less of a constraint for pollination in cleistogamic compared to chasmogamic species [227, 234]. Some crop species still have both cleistogamic and chasmogamic varieties [232, 235, 236]. Cleistogamic rice varieties were shown to be more tolerant to heat stress at flowering compared to non-cleistogamic lines [237]. However, recalcitrant pollen (e.g. maize) can lose water quickly, especially at low air humidity [238] and many cleistogamic crop species (e.g. cereals, legumes, Solanaceae) have well-documented pollen sterility problems. These prob‐ lems occur when plants experience stress at the young microspore stage or anthesis [25, 115, 191, 239–241]. Sterility in these cases may be inflicted earlier in development and may not be due to interference with pollen presentation and dispersal [242, 243]. This may indicate that cleistogamy *per se* may help avoiding pollen dispersal, but it may not offer protection against abiotic stresses that occur at other periods of flowering. Genetic manipulations and hybrid breeding in crop species have sparked renewed interest in controlling the breeding system of some crop species [231, 244–246]. Some progress has been made in recent years to identify the genes associated with the cleistogamy trait and flower opening in rice, wheat and barley [246– 249]. This research will lead to a better understanding of the genetic basis of cleistogamy and chasmogamy and the implications for abiotic stress tolerance in crop plants.

#### **9.3. Pollen size, shape and anther dehiscence**

The size of mature pollen grains at dispersal varies from less than 15 to 200 μm in diameter, with an average size of 70–100 μm in the desiccated state. The variation in pollen size has been related to the stigma size [250] and does not always correlate with water content (Table 1) [160]. Pollen grain volume increases progressively from the young microspore stage to maturity but is generally restricted by available locular space and the type of pollen dispersal unit in different species [168, 251]. The dehydration process in orthodox pollen leads to a change in shape and size of pollen grains and the harmomegathic properties of the cell wall play an important role in this process (Figure 4; Table 6) [154]. Recalcitrant pollen do not have furrows to facilitate mechanical folding of the cell wall in response to dehydration and pollen remain spherical (Figure 4; Table 6).

Pollen release from the anther requires thickening of the secondary wall of the endothecial layer (= mechanical layer) and dehydration of the epidermis [163, 252–254] (Figure 1). Dehis‐ cence mutants in Arabidopsis affect secondary wall thickening and cause male sterility; these mutants were shown to affect transcription factor genes *MYB26, NST1* and *NST2* [255–257]. Secondary cell wall thickening can also control temporary re-closure of the anther during rainy or misty weather [258, 259]. Dehydration of the epidermis is associated with increased abscisic acid (ABA) levels [260] and induction of dehydrin-like proteins [261]. Aquaporins regulate the movement of water during anther opening [262, 263]. Cells of the inter-locular septum are ruptured as a result of PCD, causing the joining of both locules of one theca – see Figures in Keijzer CJ [171] and Bonner LJ and Dickinson HG [264]. The locule volume increases and absorption of the locular fluid is accelerated [220, 265–268]. The locular content is re-distributed to other plant parts via the elongating anther filament [160, 221] and aquaporins may facilitate the movement of water through the anther wall membranes [262]. A cell death response in the stomium then causes the anther to open and pollen grains dehisce with the help of tension caused by secondary wall thickening [253]. Depending on the plant species, the stomium can rupture completely (from the top of the anther to the base), partially, or form pores for pollen dispersal [266, 267, 269]. Plant hormones regulating senescence and cell death such as auxin, jasmonic acid and ethylene play a role in anther opening and pollen dehiscence [252, 270– 273]. The elongation of the anther filament in some plant species is required to expose the anthers from the flower to facilitate dispersal (Table 5) [274].

species (e.g. wheat, barley, rice), cleistogamic breeding systems may have been selected during domestication to limit gene flow and preserve preferred gene combinations [229–233]. The absence of pollen presentation in cleistogamic self-pollinating plants is thought to be a protection against abiotic stresses such as drought and heat, as pollen number is considered less of a constraint for pollination in cleistogamic compared to chasmogamic species [227, 234]. Some crop species still have both cleistogamic and chasmogamic varieties [232, 235, 236]. Cleistogamic rice varieties were shown to be more tolerant to heat stress at flowering compared to non-cleistogamic lines [237]. However, recalcitrant pollen (e.g. maize) can lose water quickly, especially at low air humidity [238] and many cleistogamic crop species (e.g. cereals, legumes, Solanaceae) have well-documented pollen sterility problems. These prob‐ lems occur when plants experience stress at the young microspore stage or anthesis [25, 115, 191, 239–241]. Sterility in these cases may be inflicted earlier in development and may not be due to interference with pollen presentation and dispersal [242, 243]. This may indicate that cleistogamy *per se* may help avoiding pollen dispersal, but it may not offer protection against abiotic stresses that occur at other periods of flowering. Genetic manipulations and hybrid breeding in crop species have sparked renewed interest in controlling the breeding system of some crop species [231, 244–246]. Some progress has been made in recent years to identify the genes associated with the cleistogamy trait and flower opening in rice, wheat and barley [246– 249]. This research will lead to a better understanding of the genetic basis of cleistogamy and

chasmogamy and the implications for abiotic stress tolerance in crop plants.

The size of mature pollen grains at dispersal varies from less than 15 to 200 μm in diameter, with an average size of 70–100 μm in the desiccated state. The variation in pollen size has been related to the stigma size [250] and does not always correlate with water content (Table 1) [160]. Pollen grain volume increases progressively from the young microspore stage to maturity but is generally restricted by available locular space and the type of pollen dispersal unit in different species [168, 251]. The dehydration process in orthodox pollen leads to a change in shape and size of pollen grains and the harmomegathic properties of the cell wall play an important role in this process (Figure 4; Table 6) [154]. Recalcitrant pollen do not have furrows to facilitate mechanical folding of the cell wall in response to dehydration and pollen remain

Pollen release from the anther requires thickening of the secondary wall of the endothecial layer (= mechanical layer) and dehydration of the epidermis [163, 252–254] (Figure 1). Dehis‐ cence mutants in Arabidopsis affect secondary wall thickening and cause male sterility; these mutants were shown to affect transcription factor genes *MYB26, NST1* and *NST2* [255–257]. Secondary cell wall thickening can also control temporary re-closure of the anther during rainy or misty weather [258, 259]. Dehydration of the epidermis is associated with increased abscisic acid (ABA) levels [260] and induction of dehydrin-like proteins [261]. Aquaporins regulate the movement of water during anther opening [262, 263]. Cells of the inter-locular septum are ruptured as a result of PCD, causing the joining of both locules of one theca – see Figures in Keijzer CJ [171] and Bonner LJ and Dickinson HG [264]. The locule volume increases and

**9.3. Pollen size, shape and anther dehiscence**

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

spherical (Figure 4; Table 6).

Pollination in plants requires favourable interactions between pollen morphological factors and environmental conditions (Tables 6 and 7) [275]. The size and shape of pollen grains, together with the events in the anther wall regulating dehiscence all collaborate to determine desiccation time, pollen viability and pollination success (Tables 6 and 7). Variation in relative air humidity, together with abiotic stresses that affect relative humidity (heat, drought, cold stress), cause problems with pollen presentation, anther opening, dehiscence [276, 277] and pollen tube growth [32]. Precocious germination while still in the anther [278, 279], or while waiting for a pollinator to disperse the pollen (Figure 3E) [280, 281], is due to inappropriate levels of humidity. Plants have evolved clever species-specific adaptation mechanisms such as dehiscence at particular times of the day [282], dispersal as single pollen or aggregates [168, 283], active dispersal by explosive forces rupturing the anther (e.g. *Ricinus communis*) and interaction with grooming insects [284, 285].



(\*): This phase is absent when pollen leaves the anther when it opens (e.g. Poaceae) or is launched from the anther (e.g. castor bean)

**Table 7.** Stages of male gametophyte development in angiosperms and processes affected by abiotic stresses.

#### **9.4. Breeding systems and pollen:ovule ratio**

The pollen:ovule ratio (P/O) has traditionally been used as a rough estimator of plant breeding systems (Cruden 2000), but little is known about the effect of environmental stresses on this ratio. When pollen is dispersed in aggregates of hundreds of grains (e.g. massulate orchids), the locular space is restricted and limited locular fluid limits nutrition and volume increase [34]. Changes in pollen volume can be measured under optimal or stressed conditions [28, 286]. Pollen dispersed as aggregates provides greater pollination success when the ovary contains multiple ovules [168, 287] and water loss during presentation and dispersal under heat and drought conditions affects only the externally exposed pollen grains and not the internal ones. To improve pollination success, some plants produce different types of pollen (different size, shape, colour, carbohydrate and water content) in one flower. One type, fecundative pollen, is fertile and able to emit the pollen tube and fertilise, while the other type is sterile nutritive pollen that serves as a reward for pollinators who – at the same time – get dusted with fecundative pollen [288]. The flower morphology can affect accessibility of pollen by different pollinators. Self-incompatible dimorphic *Primula* species have two different flower types with reciprocal anther and style length, producing pollen with different water content depending on the position and exposure of the anthers with respect to the corolla tube [289, 290]. Three flower types, producing three types of pollen grains, occur in trimorphic species (e.g. *Lythrum salicaria*) [289, 291]. The differences in flower morphology result in non-random mating patterns in plant populations and may play an important role in pollinator selection and adapatation to different environments [292, 293].
