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. 2011 Apr 12;107(8):1421–1426. doi: 10.1093/aob/mcr074

Successive microsporogenesis affects pollen aperture pattern in the tam mutant of Arabidopsis thaliana

B Albert 1,2,*, C Raquin 1,2, M Prigent 2,3, S Nadot 1,2, F Brisset 4, M Yang 5, A Ressayre 1,2,6
1Univ. Paris-Sud, Laboratoire Ecologie Systématique et Evolution, UMR8079, Orsay, F-91405, France
2CNRS, Orsay, F-91405, France
3Univ. Paris-Sud, Institut de Génétique et Microbiologie, UMR 8621, Orsay, F-91405, France
4Univ. Paris-Sud, ICMMO, UMR8182 CNRS, Orsay, F-91405, France
5Department of Botany, Oklahoma State University, 104 Life Sciences East, Stillwater, OK 74078 –3013, USA
6INRA/Univ. Paris-Sud, UMR de de Génétique Végétale, Gif sur Yvette, F-91190, France
*

For correspondence. E-mail: beatrice.albert@u-psud.fr

Received 2010 Dec 16; Revised 2011 Feb 1; Accepted 2011 Feb 14; Issue date 2011 Jun.

© The Author 2011. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com
PMCID: PMC3101141 PMID: 21489970

Abstract

Background and Aims

The tam (tardy asynchronous meiosis) mutant of Arabidopsis thaliana, which exhibits a modified cytokinesis with a switch from simultaneous to successive cytokinesis, was used to perform a direct test of the implication of cytokinesis in aperture-pattern ontogeny of angiosperm pollen grains. The aperture pattern corresponds to the number and arrangement of apertures (areas of the pollen wall permitting pollen tube germination) on the surface of the pollen grain.

Methods

A comparative analysis of meiosis and aperture distribution was performed in two mutant strains of arabidopsis: quartet and quartet-tam.

Key Results

While the number of apertures is not affected in the quartet-tam mutant, the arrangement of the three apertures is modified compared with the quartet, resulting in a different aperture pattern.

Conclusions

These results directly demonstrate the relationship between the type of sporocytic cytokinesis and pollen aperture-pattern ontogeny.

Keywords: Cytokinesis, microsporogenesis, pollen, aperture pattern, A-type cyclin, tam, tardy asynchronous meiosis, Arabidopsis thaliana

INTRODUCTION

Microsporogenesis or male meiosis is the earliest step in pollen ontogeny. It consists of nuclear divisions associated with cytoplasmic divisions or cytokinesis. This process starts with microsporocytes or pollen mother cells enclosed in a callose envelope within which meiosis takes place. Cytokinesis takes place through the formation of intersporal walls composed of callose. Once meiosis is completed, the four microspores form a tetrad embedded within the callose wall of the pollen mother cell, until the callose is digested by an enzyme called callase. In most species, apertures are already visible at the late tetrad stage, suggesting that aperture pattern (shape, number and distribution of apertures on the pollen grain surface within the tetrad) is determined during microsporogenesis.

Two basic types of cytokinesis occur in angiosperms, successive or simultaneous (only a few cases of intermediate cytokinesis have been recorded). In successive cytokinesis, the cytoplasm is successively partitioned after each meiotic division. A dyad stage is thus observed which consists of two cells embedded within the pollen mother cell wall and separated by a callose wall. In simultaneous cytokinesis, intersporal callose walls are formed only after both nuclear divisions have taken place. These two types of cytokinesis result in different tetrad morphologies. Tetrads obtained through successive cytokinesis can be tetragonal, decussate, T-shaped, Z-shaped and linear, whereas tetrads resulting from simultaneous cytokinesis can be tetrahedral, rhomboidal, tetragonal and decussate.

Most eudicots have a simultaneous cytokinesis; only a few exceptions where cytokinesis is successive have been recorded so far (Furness, 2008). Exceptions occur in the following orders: Ranunculales (Furness, 2008), Proteales (Blackmore and Barnes, 1995), Malpighiales, Malvales (Furness, 2008) and Gentianales (Furness, 2008). In Nelumbo (Proteales), the cytokinesis was described as successive by Kreunen and Osborn (1999), but Banks et al. (2007) demonstrated that it is actually of the simultaneous type. Furness (2008) shows that successive cytokinesis has arisen at least six times independently throughout eudicots. In monocots, cytokinesis is predominantly successive but the simultaneous type is also present and has evolved several times independently (Furness and Rudall, 1999, 2000; Furness et al., 2002; Nadot et al., 2008).

Several authors have suggested that the type of cytokinesis is implicated in pollen aperture ontogeny. Wodehouse (1935) followed by Blackmore and Crane (1988) suggested that the spatial information determining aperture locations within the tetrad is provided by the last contact points persisting at the end of cytokinesis between the cytoplasms of the future microspores. Ressayre et al. (2002) have proposed a model that predicts these last contact points between microspores. This model is based on the interaction between three meiotic characters: the type of cytokinesis, the tetrad shape (which results from the orientation of the second meiotic axes) and the intersporal callose wall formation (Fig. 1). Cytokinesis type (successive/simultaneous) and tetrad shape (tetragonal/rhomboidal/tetrahedral) determine the number and the spatial arrangement of cleavage walls among nuclei, and the timing of callose wall formation (following each meiotic division in successive cell division, or after all meiotic divisions have been completed in the simultaneous type). The mode of cleavage wall formation (centrifugal/centripetal), associated with the number and spatial distribution of cleavage walls, and with the timing of cleavage wall formation, determines the areas where cytokinesis will be completed. Ressayre et al. (2002) furthermore proposed that the apertures are found at these last points of contact as suggested by Wodehouse (1935) (grouped apertures) or are oriented toward these last points (polar apertures). This theoretical model is validated in some cases (see Ressayre et al., 2002).

Fig. 1.

Fig. 1.

The four components implicated in aperture-pattern ontogeny. (A) Cytokinesis type. A characteristic dyad stage is observed in successive cytokinesis. Nuclei are represented by small blue circles, callose walls by black bars. (B) Callose deposition. Callose wall formation is illustrated in a tetragonal tetrad (circle with cleavage walls inside). The callose is represented by thick grey lines. Arrows indicate the progression of callose deposition. Red circles represent the location of the last points where callose is deposited between the microspores; the position of these points depends on the way the callose walls are formed. (B-a) Callose deposition starts from the periphery of the pollen mother cell and progresses centripetally toward the centre of microsporocyte. (B-b) Callose deposition starts from the periphery of each cleavage plane and progresses centripetally towards the centre of the cleavage plane. B-c) Callose deposition starts from the centre of each cleavage plane and progresses centrifugally. (B-d) Callose deposition starts from the centre of the microsporocyte and progresses centrifugally. (C) The shape of the tetrad. Blue circles represent pollen grains; the cleavages walls are represented in black and numbered. (C-a) Tetragonal tetrad displays three (in successive cytokinesis) or four cleavage walls (in simultaneous cytokinesis). (C-b) Rhomboidal tetrad displays five cleavage walls. (C-c) Tetrahedral tetrad displays six cleavage walls. (D) Example of aperture distribution within tetrad. (D-a) Last points of callose deposition (red points) within a tetragonal tetrad. In this example the callose deposition corresponds to a centrifugal callose deposition within cleavage planes. (D-b) Polar aperture distribution. The pollen grains are monosulcate with the sulcus oriented toward the red points. (D-c) Grouped aperture distribution. The pollen grains are diporate and the apertures are directly located where the red points are.

In this study, the tam (tardy asynchronous meiosis) mutant of Arabidopsis thaliana, which displays a switch from simultaneous to successive cytokinesis (Magnard et al., 2001), was used to perform a direct test of the implication of cytokinesis type on pollen aperture-pattern determination. It is shown that a modification of the cytokinesis type has an impact on aperture arrangement within the tetrad but does not affect the number of apertures. The aperture pattern is altered but not the tricolpate aperture type.

MATERIALS AND METHODS

Plant material and growth conditions

The quartet-1 (qrt1) strain of Arabidopsis thaliana was kindly provided by the Nottingham Arabidopsis Stock Centre. This mutant presents a failure of pectin degradation in the pollen mother cell, which results in the pollen grain being released in a tetrad (Preuss et al., 1994; Rhee and Somerville, 1998). The tam mutant has a point mutation in an A-type cyclin (Wang et al., 2004) and is slowed in the progression of male meiosis, resulting in the formation of intermediary dyads before the formation of tetrads (Magnard et al., 2001). This mutant is temperature sensitive and the tam phenotype is much more pronounced at 27 °C than at 20 °C or 15 °C. The double quartet-tam mutant was constructed by crossing both tam and quartet mutants. Quartet and quartet-tam plants were grown under controlled conditions at 20 °C day and 15 °C night under 14 h light per day for 3–4 weeks until they reached the reproductive stage. Then the plants were transferred to 27 °C and 1 week later flower-bud collection started.

Microscopy

Fresh flower buds were collected at various developmental stages. Several flower buds per individual and several stamens per bud were sampled and studied for each developmental stage. The anthers were dissected out, immediately squashed and mounted in aniline blue to which 15 % glycerol was added, following a modified protocol from Arens (1949). With this method, callose becomes fluorescent when UV illuminated and observed with a blue filter (excitation at 345, emission at 425 nm long pass). The last contact points between microspores were observed using a special DAPI (diamidino-2-phenylindole) preparation. Fixation of anthers was achieved by squashing them on a slide in freshly prepared solution of 8 % formaldehyde in buffer A {50 mmol L−1 PIPES, 5 mmol L−1 EGTA [ethylene glycol bis (2-aminoethyl ether)-N, N, N9N9-tetraacetic acid], 5 mmol L−1 MgSO4, pH 5–6·9; Traas et al., 1989}. The slides were stored for 2 d at –80 °C, washed twice with buffer A, and incubated for 15 min with a DAPI solution and washed twice again with buffer A. The slides were mounted in citifluor. Preparations were observed using epifluorescence with a blue filter. A field emission gun scanning electron microscope was used to observe dehydrated tetrads of pollen grains. The experimental conditions were chosen to avoid both the use of a metallic coating and deep penetration in the organic sample. To achieve that, a 1-kV high voltage was used with a low current probe, permitting observation without fatal charging effect and allowing a detailed sample surface view.

RESULTS

To understand the effect of the tam mutation on the pollen aperture pattern, microsporogenesis was observed in two mutant strains of arabidopsis: quartet and quartet-tam. The Qrt mutation provided an easy means to visualize the arrangement of apertures on pollen grains assembled in tetrads, i.e. the aperture pattern.

Aperture pattern

Pollen grains were tri-aperturate in both the quartet (Fig. 2C) and quartet-tam strains (Fig. 2F; a low frequency of abnormal pollen grains was found, e.g. in Fig. 2E), but the aperture patterns were different between the two strains (Fig. 2A–F). In the quartet strain, tetrads were tetrahedral, and each of the three apertures of a pollen grain was in contact with one aperture of each of the three other pollen grains of the tetrad. This corresponds to the classical Fischer arrangement of apertures (Fischer, 1890). In the quartet-tam strain, tetrads were mostly tetragonal (Fig. 2D, E), although decussate (Fig. 2F), linear, T-shaped and Z-shaped tetrads were also observed. Each of the three apertures of a pollen grain was in contact with the aperture of only one pollen grain among the three others (Fig. 2D–F).

Fig. 2.

Fig. 2.

(A–C) Pollen tetrad in quartet strain. Pollen grains are three aperturate – each of the three apertures of a pollen grain is associated with an aperture of another pollen grain. Apertures of a pollen grain are in contact with apertures of the three other pollen grains. Tetrads are tetrahedral. (C-b) Scheme of the tetrad shown in (C-a); big circles represent the microspores, triangles represent apertures. (D–F) Pollen tetrad in quartet-tam strain. Three aperturate pollen grains are associated two by two within the tetrad. All the three apertures of a pollen grain are associated with the three apertures of only one another pollen grain. (D) Tetragonal tetrad. (E) Tetragonal tetrad showing a pollen grain with an abnormal aperture (the upper right). (F-a, -b, -c) Different focal planes on a decussate tetrad; arrows show apertures. (F-d) Scheme of the tetrad shown in (F-a, -b, -c), big circles represent the microspores; black bars represent apertures that are seen by looking from the view of the tetrad in (F-a); grey ones those which are hidden. (A), (D) and (E) are scanning electron micrographs; (B), (C) and (F) are stained with aniline blue. Scale bars = 10 μm.

Microsporogenesis

In the quartet strain, cytokinesis was simultaneous (Fig. 3A). Callose wall formation began at the periphery of each cleavage wall and progressed centripetally (Fig. 3A). Each microspore had one last contact point with the three others (Fig. 3B). This last contact point corresponded to the place where intersporal callose wall formation was completed (Fig. 3C). The future apertures appeared to locate precisely at these last points of callose deposition. In the quartet-tam strain, cytokinesis was successive (Fig. 3D, E). The two cytoplasmic divisions were achieved by the formation of callose walls which first formed at the periphery of the cleavage sites and progressed centripetally (Fig. 3D, F). Three last contact points between microspores were observed at the end of the second cytoplasmic division (Fig. 3H). These last contact points correspond to the last points where intersporal wall formation was completed (Fig. 3I). Differing from the situation in the quartet strain in which each microspore was in contact with the three others, here the three last contact points were between two microspores of each pair within the tetrad.

Fig. 3.

Fig. 3.

(A–C) Microsporogenesis in quartet strain. (A) Centripetal cell plate formation within a cleavage plane; callose deposition started from the border of the tetrad and in the centre (arrows). (B) Two focal planes of a tetrad at the end of cytokinesis; the three last contact points between microspores are indicated by arrows. (C) Tetrahedral tetrad after the completion of cell plate formation. (D–I) Microsporogenesis in the quartet-tam strain. (D) Centripetal cell plate formation within a cleavage plane after the first nuclear division. (E) Dyad. (F, G) Centripetal cell plate formation after the second nuclear division in a decussate tetrad (F) and in a tetragonal tetrad (G). (H-a,b) Two focal planes of a tetrad at the end of cytokinesis showing the three last contact points between two microspores (arrows); it is not possible to see the other two microspores (on left) very well. (I) Tetragonal tetrad after the completion of cell plate formation. All are stained with aniline blue. Scale bars = 10 μm.

DISCUSSION

The first report of the implication of a cytokinetic mutant in the determination of the aperture pattern is the tes/stud mutants (Hulskamp et al., 1997; Spielman et al., 1997; Yang et al., 2003). These mutants present as a primary defect a failure in cytokinesis: the four meiotic nuclei remain within the same cytoplasm. These mutants display extra apertures in abnormal orientations on the pollen wall. The absence of cytoplasm partitioning in tes/stud mutants has multiple consequences, which make it difficult to identify clearly which factors are directly implicated in aperture modification. In this work, two strains differing only in the type of cytokinesis during microsporogenesis were compared, making it possible to identify more clearly the impact of cytokinesis on aperture-pattern determination. Quartet and quartet-tam present similarities in the way microsporogenesis is achieved, which includes the centripetal deposition of intersporal callose walls within each cleavage plane, the tricolpate aperture type and the grouped position of apertures within the tetrad (see Fig. 1D-c). The aperture pattern is, however, different between the two strains. Since the only difference in the unfolding of microsporogenesis is the type of cytokinesis, which is simultaneous in quartet and successive in quartet-tam, it demonstrates that the way in which cytokinesis takes place specifies the pollen aperture patterning.

In quartet and quartet-tam, the apertures are located at the last points of contact between microspores, which correspond to the points where intersporal callose walls are lastly formed. In particular, in the quartet strain, each of the three apertures of a pollen grain is in contact with one aperture of another pollen grain (meaning that each aperture of a pollen grain is in contact with the three other pollen grains) whereas in the quartet-tam mutant, the apertures of a pollen grain are in contact with the apertures of only one of the other three pollen grains. In these two strains the apertures are found at the last contact points between microspores as already described for other eudicots (Wodehouse, 1935; Huynh, 1968; Blackmore and Crane, 1988; Ressayre et al., 1998).

It is hypothesized that the locations of the last contact points between microspores are determined by three factors, namely the type of cytokinesis, the mode of intersporal callose walls formation and the tetrad shape (Albert et al., 2010), following the ontogenetic model of Ressayre et al. (2002). The distribution of the last contact points among microspores observed in the quartet mutant is in agreement with the predictions of this model. In particular, when intersporal callose wall formation is centripetal within each cleavage plane, and the tetrad shape is tetrahedral, the last points of callose deposition are expected to be located in the centre of each cleavage plane. This does not hold true for the quartet-tam mutant. According to the model of Ressayre et al. (2002), when intersporal callose wall formation is centripetal within each cleavage plane, and when the tetrad shape is tetragonal (resulting from successive cytokinesis), a single last contact point is expected to be located in the centre of each of the last two cleavage planes formed after the second meiotic division (Fig. 1B). In quartet-tam, three last contact points were observed in each of the last two cleavage planes, instead of one in the centre of each plane. In spite of careful and thorough observations, it is still unclear how these last contact points are formed. One hypothesis is that intersporal wall formation is not totally synchronous, resulting in more than one last contact point between the two microspores. Alternatively, the cytoskeleton that is supposedly responsible for the cell wall formation may inherently possess a tripartite structure that is reoriented in the mutant to simultaneously result in the three last contact points between the microspores.

Successive cytokinesis was always recorded to be associated with centrifugal intersporal callose wall formation (reviewed by Nadot et al., 2008). In addition to the main result of the present study – the demonstration that the type of cytokinesis has an impact on aperture-pattern determination – it should be noted that the quartet-tam is the first report of successive cytokinesis associated with centripetal intersporal callose wall formation.

ACKNOWLEDGEMENTS

We are grateful to Julie Sannier, Lionel Saunois and Fereol Braud.

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