Variation of microsporogenesis in monocots producing monosulcate pollen grains

Abstract

Background and Aims

Microsporogenesis leading to monosulcate pollen grains has already been described for a wide range of monocot species. However, a detailed study of additional callose deposition after the completion of the cleavage walls has been neglected so far. The study of additional callose deposition in monosulcate pollen grain has gained importance since a correlation between additional callose deposition and aperture location has recently been revealed.

Methods

Microsporogenesis is described for 30 species belonging to eight families of the monocots: Acoraceae, Amaryllidaceae, Alstroemeriaceae, Asparagaceae, Butomaceae, Commelinaceae, Liliaceae and Xanthorrhoeaceae.

Key Results

Five different microsporogenesis pathways are associated with monosulcate pollen grain. They differ in the type of cytokinesis, tetrad shape, and the presence and shape of additional callose deposition. Four of them present additional callose deposition.

Conclusions

In all these different microsporogenesis pathways, aperture location seems to be linked to the last point of callose deposition.

INTRODUCTION

The pollen grain is the male gametophyte of flowering plants. Its morphology is highly diverse, from the composition and structure of its wall to its ornamentation and general shape. Apertures, which are areas where the exine (the external layer of the pollen grain wall) is absent or greatly reduced, represent one of the striking variable features of pollen grains (Erdtman, 1952). Apertures vary in shape, number and location, a combination of features defined as the aperture pattern. They are usually in the shape of a furrow or a pore (Erdtman, 1952), and their number ranges from one to several (up to 100 in very rare cases). Sometimes no aperture is visible, either because none is present, or because the apertural zone covers the whole of the pollen grain surface (Furness, and Rudall, 2000a). Apertures play a crucial role in pollen reproductive success via germination and osmotic exchange capacities.

The variation in aperture pattern is not randomly distributed across angiosperms. The monosulcate type (one furrow in a distal position) is the most common aperture type in monocots and basal angiosperms (Furness, and Rudall, 1999, 2000b), and is considered as the ancestral condition for monocots, and also for flowering plants as a whole (Walker and Doyle, 1975; Doyle, 2005; Nadot et al., 2008). The most frequent pollen type in the eudicot clade is the tricolpate type (three furrows distributed orthogonally to the equator of the pollen grain), which is the only known synapomorphy of this huge clade (Donoghue and Doyle, 1989).

The diversity of pollen morphology in angiosperms is well documented, thanks to the work of Erdtman (Erdtman, 1952), and accurate descriptions are now available online (see Paldat, http://www.paldat.org/). However, the developmental bases of this diversity are still poorly understood. Wodehouse (1935) was the first to propose a hypothesis on aperture pattern determination. He suggested that the last contact points between the microspores in the late tetrad stage determines the position of the apertures on the pollen grain surface. Further, Ressayre et al. (2002) proposed that these last contact points between the microspores are determined by the last points of callose deposition during intersporal (or cleavage) wall formation. They suggested that the type of cytokinesis (successive or simultaneous), intersporal wall formation (centrifugal or centripetal) and tetrad shape (tetrahedral, tetragonal/decussate or rhomboidal) are strongly involved in determining these last points of contact. They proposed that equatorial apertures are localized at these last points whereas distal apertures are oriented toward these last contact points. Waterkeyn and Bienfait (1970) and Blackmore and Barnes (1987) first reported the existence of a link between callose deposition and aperture location. Since then, additional callose deposition (after completion of the callosic intersporal walls) has been studied in detail in several species (Ressayre, 2001; Ressayre et al., 2005; Albert and Nadot, 2010; Albert et al., 2010, 2011). These studies all show that there is a correlation between the last points of additional callose deposition and aperture location.

The ontogeny of monosulcate pollen grains has been examined in several families of the monocots, more or less representing the taxonomic diversity of this clade (Furness and Rudall, 1999, 2000b; Penet et al., 2005; Nadot et al., 2006; Sannier et al., 2006). These studies have revealed that monosulcate pollen grains can be obtained through several developmental pathways. Variation in these pathways concerns the type of cytokinesis (successive or simultaneous), the way in which the cleavage walls are formed (centrifugal or centripetal) and the tetrad shape (tetrahedral, tetragonal, decussate or rhomboidal). The detailed study of additional callose deposition after the completion of the cleavage walls has been neglected so far. We report here five different developmental pathways associated with monosulcate pollen grain; four are new microsporogenesis pathways and one has already been described, but we record some new species presenting this pathway (Penet et al., 2005; Sannier et al., 2006; Nadot et al., 2008). These new developmental pathways present additional callose deposition after formation of the cleavage walls. Microsporogenesis is described for 30 species belonging to eight families of the monocots (Acoraceae, Amaryllidaceae, Alstroemeriaceae, Asparagaceae, Butomaceae, Commelinaceae, Liliaceae and Xanthorrhoeaceae).

MATERIALS AND METHODS

Plant material

Danae racemosa Moench, Fritillaria meleagris L., Hemerocallis fulva L. and Polygonatum odoratum (Mill.) Druce were collected in the Parc Botanique de Launay (Orsay, France). Aloe jucunda Reynolds, Asphodeline lutea Rchb., Cordyline fruticosa (L). A.Chev., Cyanotis pilosa Schult.f., Gasteria pillansii var. ernesti-ruschii (Dinter Von Poellnitz) van Jaarsv., Haworthia cooperi Baker, Hosta sieboldiana (Hook.) Engl., Ledebouria concolor (Baker) Jessop and Ophiopogon japonicus Ker Gawl. were sampled from the Jardin Botanique de la Ville de Paris (Paris, France). Narcissus pseudonarcissus L., Puschkinia scilloides Adams and Tulipa clusiana Redouté were bought from a commercial firm. Acorus calamus L., Agapanthus africanus Hoffmanns, Alstroemeria aurantiaca D.Don, Butomus umbellatus L., Camassia leichtlinii (Baker) S.Watson, Convallaria majalis L., Kniphofia rufa Baker, Ornithogalum candicans (Baker) J.C.Manning & Goldblatt, Leucojum aestivum L., Lilium henryi Baker, Liriope muscari L.H.Bailey, Maianthemum bifolium (L.) F.W.Schmidt and Scilla luciliae (Boiss.) Speta were collected from the Muséum National d'Histoire Naturelle (Paris, France). Veltheimia bracteata Harv. ex Baker was collected from the greenhouses of the Muséum National d'Histoire Naturelle (Chèvreloup, France).

Microscopy

Fresh flower buds were collected from the different species at various developmental stages. Several flower buds and several stamens per bud were sampled and observed for each developmental stage. The anthers were dissected out, immediately squashed and mounted in a solution of aniline blue (1 mm aniline blue, 80 mm K₃PO₄, 15 % glycerol; adapted from Arens, 1949). With this method, callose becomes fluorescent when UV illuminated [DAPI (4,6'-diamidino-2-phenylindole) filter]. We recorded the type of cytokinesis, the progression of intersporal callose wall formation, the deposition of additional callose, the resulting tetrad shape and the arrangement of apertures within tetrads in the studied species.

RESULTS

In all the species studied, cytokinesis is successive (Fig. 1A, B), as shown by the presence of a dyad stage, except in A. jucunda, H. cooperi, G. pillansii, A. lutea and K. rufa where cytokinesis is simultaneous (Fig. 1M), and H. fulva which present intermediate cytokinesis (Fig. 1H). The formation of the cleavage walls is always achieved by cell plates developing centrifugally (Fig. 1A, B, H, M). Tetrads are mostly tetragonal (Fig. 1C, E, G, I–L), although decussate tetrads are also observed (Fig. 1D). Aloe jucunda, H. cooperi, G. pillansii, A. lutea and K. rufa produce asymmetric tetrahedral tetrads (Fig. 1N). Hemerocallis fulva presents tetragonal and decussate tetrads associated with a few cases of tetrahedral and rhomboidal tetrads. In N. pseudonarcissus (Asparagales-Amaryllidaceae), C. leichtlinii (Asparagales-Asparagaceae), C. pilosa (Commelinales-Commelinaceae), A. calamus (Acorales-Acoraceae), A. aurantiaca (Liliales-Alstroemeriaceae), F. meleagris and T. clusiana (Liliales-Liliaceae), intersporal walls consisted of bare callosic cell plates. No additional callose deposits were observed on the cell plates after their formation. In B. umbellatus (Alismatales-Butomaceae), A. africanus and L. aestivum (Asparagales-Amaryllidaceae), S. luciliae, C. majalis, C. fruticosa, O. candicans, H. sieboldiana, P. scilloides, L. concolor, L. muscari, M. bifolium, O. japonicus, P. odoratum and V. bracteata (Asparagales-Asparagaceae), and L. henryi (Liliales-Liliaceae) additional callose deposits were observed on the upper and lower focus of the tetrad, visible only once the formation of the cleavage walls was achieved (Fig. 1G). In D. racemosa (Asparagales-Asparagaceae), two kinds of additional callose deposits were observed. The first consists of thickenings on the upper and lower focus of the tetrad (Fig. 1J) and the second has the shape of a sulcus, located at the place where the aperture is later formed (Fig. 1K, L). In H. fulva (Asparagales-Xanthorrhoeaceae), an elongated patch in the shape of a sulcus of callose located at the place where the pollen grain sulcus is later formed was observed on the tetrad wall, only after the completion of the cleavage walls (Fig. 1I). In Aloe jucunda, H. cooperi and G. pillansii (Asparagales-Asparagaceae), A. lutea and K. rufa (Asparagales-Xanthorrhoeaceae) an additional callose deposit is present at the intersection between the cell plates and the outer wall of the tetrad (Fig. 1O). All the studied species present monosulcate pollen grains (Fig. 1F) arranged in a tetragonal tetrad (or decussate) as shown in Fig. 1E, except A. jucunda, H. cooperi, G. pillansii, A. lutea and K. rufa where the monosulcate pollen grains are arranged in tetrahedral tetrad (Fig. 1P).

Fig. 1.

Microsporogenesis. (A–F) Cyanotis pilosa, (G) Puschkinia scilloides, (H, I) Hemerocallis fulva, (J–L) Danae racemosa and (M–P) Haworthia cooperi. (A, B) Successive cytokinesis with formation of centrifugal cleavage walls. (C) Tetragonal tetrad. (D) Decussate tetrad. (E) Pollen grains within a tetrad exhibiting a single distal sulcus (arrow). (F) Monosulcate pollen grain. (G) Lower (1), middle (2) and upper (3) focus on a tetragonal tetrad with additional callose deposits. On the lower and upper focus, thick deposits are observed at the intersection between the cleavage walls (arrow). On the middle focus (2, cross-section of the tetrad), no additional callose deposit is observed. (H) Simultaneous cytokinesis with formation of centrifugal cleavage walls. (I) Additional callose deposit located at the place where the sulcus will be found later (arrow). (J) Lower (1), middle (2) and upper (3) focus on a tetragonal tetrad with additional callose deposits. On the lower and upper focus, thick deposits are observed at the intersection between the cleavage walls (arrow). On the middle focus (2, cross-section of the tetrad), no additional callose deposit is observed. (K, L) Tetrad at a later stage than (J), with a new additional callose deposit located at the place where the sulcus will be found later (arrow). (M) Simultaneous cytokinesis with formation of centrifugal cleavage walls. (N) Asymmetric tetrahedral tetrad. (O) Lower (1), middle (2) and upper (3) focus on a tetrahedral tetrad with additional callose deposits at the intersection between the cleavage walls and the outer wall of the tetrad. (P) Lower (1) and upper (2) focus on a tetrahedral tetrad with monosulcate pollen grains. Scale bars = 10 μm.

DISCUSSION

We describe here five different microsporogenesis pathways associated with formation of centrifugal cleavage walls (Table 1). These pathways differ in type of cytokinesis, tetrad shape and callose deposition. The first one presents successive cytokinesis, tetragonal/decussate tetrads and no additional callose deposition. The second one involves successive cytokinesis, tetragonal/decussate tetrads and additional callose deposits on the upper and lower focus of the tetrad. The third one presents successive cytokinesis, tetragonal/decussate tetrads and additional callose deposits on the upper and lower focus of the tetrad followed by another deposit in the shape of a sulcus. The fourth one involves intermediate cytokinesis, tetragonal/decussate and a few cases of tetrahedral and rhomboidal tetrads, and an additional callose deposit in the shape of a sulcus. In the last one, cytokinesis is simultaneous, tetrads are asymmetric tetrahedral, and additional callose deposits are observed at the intersection of the cell plates and the outer tetrad wall.

Table 1.

The five different pathways of microsporogenesis recorded in this study: all pathways lead to monosulcate pollen.

Cytokinesis	Tetrad shape	Additional callose deposits
Successive	Tetragonal/decussate	None
Successive	Tetragonal/decussate	On the upper and lower focus of the tetrad
Successive	Tetragonal/Decussate	On the upper and lower focus of the tetrad + sulcus-like on each microspore
Intermediate	Tetragonal/decussate/tetrahedral/rhomboidal	Sulcus-like on each microspore
Simultaneous	Asymmetric tetrahedral	At the intersection between intersporal walls and outer tetrad wall

The type of cytokinesis observed for all species examined except H. fulva, A. jucunda, H. cooperi, G. pillansii, A. lutea and K. rufa corresponds to the most common pathway of microsporogenesis described so far in monocots. In this pathway, cytokinesis is successive, with intersporal wall formation achieved through cell plates developing centrifugally (Dahlgren and Clifford, 1982; Furness and Rudall, 1999), resulting in tetrads that are generally tetragonal and decussate, occasionally linear or T-shaped. Simultaneous cytokinesis has been recorded in members of several orders of the monocots including the Asparagales, Dioscoreales, Cyperales and Arecales (Furness and Rudall, 1999; Penet et al., 2005; Sannier et al., 2006). Intraindividual variation in the type of cytokinesis has even been described in the Arecales (Sannier et al., 2006) where a few cases of simultaneous cytokinesis occurring together with successive cytokinesis within a single individual were found. When cytokinesis is simultaneous, the tetrad shape can be variable, including tetrahedral (regular or irregular), tetragonal, decussate or rhomboidal. Intersporal wall formation is generally centripetal, but cases of centrifugal formation have been recorded. The only cases of simultaneous cytokinesis found in our sample of species occur in A. jucunda, H. cooperi, G. pillansii, A. lutea and K. rufa in which intersporal wall formation is centrifugal and results in asymmetric tetrahedral tetrads. We also confirmed one case of intermediate cytokinesis in H. fulva (Penet et al., 2005). In this species, intersporal wall formation is achieved through centrifugally developing cell plates, like in the species where cytokinesis is successive, and results in tetragonal tetrads (Penet et al., 2005). The other case of intermediate cytokinesis described is in Tritonia securigera associated with centripetal cell wall formation and tetragonal tetrads (Penet et al., 2005).

One major novelty compared here with previous descriptions of microsporogenesis in the Asparagales (and even in monocots as a whole) is the fine description of the presence of additional callose deposits in species producing monosulcate pollen grain. In a few species (A. calamus, A. aurantiaca, C. leichtlinii, C. pilosa, F. meleagris, N. pseudonarcissus and T. clusiana), the cell plates remain bare until the very end of the tetrad stage. In contrast, in all other species additional callose deposits are observed. Interestingly, the presence or absence of such additional callose deposits on the intersporal walls does not seem to follow any particular systematic pattern, since both situations are found in Amaryllidaceae, Asparagaceae and Liliaceae. The fact that among the 17 species of the Asparagaceae family examined, only one is devoid of additional callose deposits suggests that the absence of additional callose would result from a secondary loss rather than being the ancestral condition in the family. Two other types of additional callose deposition have been observed, each in only one species. In H. fulva (Xanthorrhoeaceae), there is an additional callose deposit in the shape of a furrow on the inner side of the outer tetrad wall. In D. racemosa (Asparagaceae), the same pattern is observed but it is preceded by a first callose deposit occurring on the upper and lower focus of the tetrad.

Although various patterns of additional callose deposition have been observed previously (Waterkeyn and Bienfait, 1970; Blackmore and Barnes, 1987; Ressayre, 2001; Ressayre et al., 2005; Albert and Nadot, 2010; Albert et al., 2010, 2011), the patterns described here have never been observed before in angiosperms. In all cases, including the descriptions presented here, the position of the callose deposits always coincides with the position of the future apertures, suggesting perhaps an implication of these deposits in the developmental process leading to the formation of apertures. In the species described here, two different situations are found. The first situation corresponds to the species presenting tetrads devoid of additional callose deposits on the cell plates at the end of microsporogenesis, or with additional thickenings of callose located at the intersection between the outer tetrad wall and the walls formed after the second meiotic division. In either case, intersporal wall formation (including additional callose deposition) is completely achieved at the places marked with red dots on Fig. 2A, which therefore correspond to the places where callose is last deposited. In this situation, the model of Ressayre et al. (2002) predicts that the sulcus of each pollen grain will be oriented in such a way that both ends of the sulcus are located precisely where intersporal wall formation is completed (Fig. 2B). Our data are in agreement with this prediction, as shown by the distribution of apertures within the tetrad. The other situation corresponds to the two cases where a ‘sulcus-like’ patch of callose is observed at the late tetrad stage and corresponds then to the last point of callose deposition. This situation had been overlooked in the model of Ressayre et al. (2002) because it had not been yet described at that time. In tetrahedral tetrads, the sulcus on the pollen grain seems to be oriented toward the additional callose deposits. The correlation found between patterns of additional callose deposition and aperture pattern in this study and in other species described previously strongly suggests, as mentioned above, a direct role for callose deposits in the determination of aperture pattern.

Fig. 2.

Relationship between patterns of callose deposition and aperture pattern. (A) Schematic representation of a tetragonal tetrad. Callosic cell plates are in grey. A first cell plate is formed centrifugally, resulting in a dyad (successive cytokinesis), and the second cleavage planes are formed by centrifugally developing cell plates (arrows simulate this centrifugal process). The red dots indicate the last points where callose will be deposited. (B) The distal sulcus of each pollen grain has both its ends oriented toward the last points of callose deposition.

The fact that different patterns of callose deposition, during and after intersporal wall formation, can all result in the same aperture pattern, also suggests that different developmental mechanisms are probably involved in aperture pattern determination. Further investigations are required to understand these mechanisms and to understand why sometimes there is callose laid at the places where the apertures are formed, whereas in other cases the apertures are oriented toward such additional callose deposits.