Protoplasma DOI 10.1007/s00709-015-0793-6
ORIGINAL ARTICLE
Ultrastructure of microsporogenesis and microgametogenesis in Brachypodium distachyon Akanksha Sharma & Mohan B. Singh & Prem L. Bhalla
Received: 16 October 2014 / Accepted: 2 March 2015 # Springer-Verlag Wien 2015
Abstract Brachypodium distachyon has emerged as a model system for forage grass and cereal grain species. Here, we report B. distachyon pollen development at the ultrastructural level. The process of microsporogenesis and microgametogenesis in B. distachyon follows the typical angiosperm pollen development sequence. Pronounced evaginations of the nuclear envelope are observed prior to meiosis, indicating active nucleocytoplasmic exchange processes. The microspore mother cells undergo meiosis and subsequent cytokinesis, forming isobilateral tetrads. Following dissolution of the callose wall and release of free and vacuolated microspores, mitotic divisions lead to the formation of mature, three-celled pollen grains. In B. distachyon, pollen wall formation begins at the tetrad stage by the formation of the exine template (primexine). The exine is tectate-columellate, comprising a foot layer and endexine. Development of the tectum and the foot layer is complete by the free microspore stage of development, with the tectum formed discontinuously. The endexine initiates in the free microspore stage but becomes compressed in mature grains. The intine layer is deposited after mitosis and comprises three layers during the mature pollen stage of development. Pore development initiates during early free microspore development stage and Brachypodium pollen has a single germination pore consisting of a slightly raised annulus surrounding a central operculum. The tapetum is of the secretory type with loss of the tapetal cell walls beginning at about the time of microsporocyte meiosis. This is the first report on ultrastructure of microsporogenesis and microgametogenesis in B. distachyon. In general,
Handling Editor: Benedikt Kost A. Sharma : M. B. Singh : P. L. Bhalla (*) Plant Molecular Biology and Biotechnology Laboratory, Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, Melbourne, VIC 3010, Australia e-mail: [email protected]
Brachypodium microsporogenesis and microgametogenesis conform to a typical grass pollen development pattern. Keywords Brachypodium distachyon . Ontogeny . Pollen . Pollen wall . Ultrastructure . Microsporogenesis . Microgametogenesis
Introduction Male reproductive development in flowering plants is initiated by the formation of stamens that house sporogenous cells, the microsporocytes. Microsporogenesis comprises the sequence of successive developmental stages from sporogenous cells to haploid unicellular microspores. During this process, the microsporocytes undergo meiotic division to generate four haploid microspores (tetrad) enclosed within a callose wall. Microgametogenesis begins at the release of haploid microspores from tetrads. The haploid microspores then undergo asymmetric division to give rise to bicellular pollen that contains a smaller generative cell enclosed within a larger vegetative cell. The generative cell further divides to give rise to the two sperm cells required for double fertilization. This study focuses on the ultrastructural changes occurring throughout the process of microsporogenesis and microgametogenesis in Brachypodium distachyon. B. distachyon, a member of the Pooideae subfamily, is a small annual grass with biological, physical and genomic attributes of a model plant for grasses and cereal crops (Draper et al. 2001; Garvin et al. 2008; Opanowicz et al. 2008; Bevan et al. 2010; Girin et al. 2014). It is endemic to the Mediterranean and Middle East and belongs to the Bcore pooid^ genera that include the majority of important temperate cereals and forage grasses, such as wheat, barley and switch grass (Draper et al. 2001).
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Deciphering the morphological events involved in microsporogenesis and microsporogenesis is critical for understanding the reproductive biology and phylogeny of flowering plants (Blackmore et al. 2007). The study of pollen development for the genus Brachypodium has revealed that it has trinucleate, uniporate, spheroidal pollen grains with granular ornamentation. The microspore cytoplasm contains variable amounts of insoluble polysaccharides and proteins at different developmental stages. Anther wall development follows the monocotyledonous-type, which is composed of an epidermal layer, an endothecial layer, one middle layer and a secretorytype tapetum (Harsant et al. 2013; Sharma et al. 2014a, b). The ultrastructural features of pollen development have been addressed in other members of the Pooideae family, such as wheat (El-Ghazaly and Jensen 1986; Mizelle et al. 1989), maize (Skvarla and Larson 1966) and sorghum (Christensen et al. 1972; Christensen and Horner 1974). In contrast, there is no study available to date on the ultrastructural features of pollen development in the genus Brachypodium. The present study integrates ultrastructural and developmental observations to document critical pollen developmental events in B. distachyon using transmission electron microscopy (TEM). We paid special attention to B. distachyon pollen wall development, as there are significant variations in the pollen wall architecture and timing of distinct developmental stages between species.
Materials and methods Fresh immature spikelets and anthers at various stages of development were collected from B. distachyon (Bd21-3) plants on different days based on the visibility of the first awn out of the flag leaf to the stage when dehisced anthers were noticeable (a period of 9 days). Spikelets and anthers were classified into groups according to the related stage of pollen development (Table 1). Their stage of development had been previously determined by examining LR White-embedded sections using light microscopy (Sharma et al. 2014a). For the transmission electron microscopy (TEM) studies, spikelets and anthers were fixed in cold 2.5 % glutaraldehyde/ 0.1 M PBS (phosphate-buffered saline), pH 7.0, for 4 h at room temperature. Following dehydration in an ethanol series, the material was then incubated in 1:1 and 1:3 ethanol:resin (LR White) mixtures for 1 day each at room temperature and finally overnight in 100 % resin on a rotating platform. Following dehydration, the spikelets and anthers were embedded in LR White, which was polymerized by incubation at 60 °C for 24–30 h. Ultrathin sections (90 nm) were cut using a Leica Ultracut® microtome and then stained with 2 % uranyl acetate for 10 min, followed by triple lead stain for 5 min (Sato 1968). The sections were observed and photographed with a Philips CM120 BioTwin transmission electron microscope.
Terminology The terminology used to describe the different exine layers of B. distachyon is defined according to Hesse et al. (2009).
Results Although microsporogenesis and microgametogenesis comprise a continuous process, to clearly describe the events occurring during pollen ontogeny in B. distachyon, the process is classified into seven developmental stages: sporogenous cell, microspore mother cell, tetrad, free microspore, vacuolated microspore, bicellular pollen and mature pollen stages. Sporogenous cell stage At this stage, the microsporocytes or the sporogenous cells were slightly polygonal and tightly packed to completely fill the locular space. The sporogenous cells were usually arranged into two rows on longitudinal sections. These cells contained large, centrally located nuclei with prominent nucleoli, exhibited thin walls and were connected to each other by plasmodesmata (Fig. 1a). A few vacuoles, plastids and mitochondria were observed in the cytoplasm of the sporogenous cells (Fig. 1a). The nuclei of these cells showed scattered and diffuse chromatin. The outer membrane of the nuclear envelope was observed to be evaginated at a few sites (Fig. 1a), possibly constituting a line of transport and exchange between the nucleus and cytoplasm. During the sporogenous cell stage, the anther wall completed its differentiation, comprising of four layers: the epidermis, the endothecium, the middle layer and the tapetum (Fig. 1b). Plasmodesmatal connections were observed between adjacent tapetal cells and between the microsporocytes and tapetal cells (Fig. 1b). The tapetal cells were rectangular in outline and possessed a single large nucleus. Their cytoplasm stained more densely, in contrast with the microspore cytoplasm. Small vacuoles, few plastids and mitochondria were observed in tapetal cytoplasm (Fig. 1b). Microspore mother cell stage At the premeiotic stage, the microspore mother cells showed a centrally located, large nucleus and nucleolus and were connected to each other by cytoplasmic connections. At this stage, numerous plastids, mitochondria and slightly bigger vacuoles were observed throughout the microsporocyte cytoplasm (Fig. 2a). Many of the mitochondria were observed to be elongated and were most likely in the process of dividing. Few plastids of the microspore mother cells were observed elongated or containing internal membranes (Fig. 2a). Just before meiotic divisions, the microsporocytes became less
Microsporogenesis and microgametogenesis in B. distachyon Table 1
Temporal developmental stages of anther and microspores of B. distachyon (Sharma et al. 2014a)
Daysa
Plant material fixed and sectioned
No. of stages identified
Stages identified
Stage of the base floret
1 2
Spikelet Spikelet
2 3
Primary parietal cell stage Pollen mother cell stage
3
Spikelet
4
4
Spikelet
3
5
Spikelet
3
6 7 8 9
Anther Anther Anther Anther
1 1 1 1
Archesporial cell stage, primary parietal cell stage Primary sporogenous cell stage, sporogenous cell stage and pollen mother cell stage Primary parietal cell stage, primary sporogenous cell stage, sporogenous cell stage and Pollen mother cell stage Sporogenous cell stage, pollen mother cell stage and tetrad stage Pollen mother cell stage, tetrad stage and free microspore stage Vacuolated microspore stage Vacuolated microspore stage Bicellular pollen stage Mature pollen stage
Pollen mother cell stage
Tetrad stage Free microspore stage – – – –
a
Day 1: when the first awn was just visible out of the flag leaf; days 2–9: subsequent days after awn visibility; day 9: anthesis, when yellow, mature anthers were clearly visible
polygonal, elliptical or irregular, and were still connected to each other. The tapetal cells undergo nuclear division and appeared binucleated at this stage and were connected to each other and to the pollen mother cells by their original plasmodesmata (Fig. 2b). At this stage, tapetal cells have a greater density of cytoplasm than in the previous stage with numerous mitochondria, plastids and few small vacuoles (Fig. 2b). Tetrad stage After meiosis, tetrads of haploid microspores randomly filled the entire space of the anther locules. Following successive
Fig. 1 TEM micrographs of longitudinal sections of spikelets at sporogenous cell stage. a Ultrastructure of sporogenous cells. Black arrows show the plasmodesmata between adjacent sporogenous cells. White arrows show the evaginations of the nuclear membrane. b Ultrastructure of anther walls at sporogenous cell stage. Arrows show
cytokinesis, numerous tetrads with isobilateral arrangement (Fig. 3a) were observed. Each tetrad enclosed four haploid microspores within an asymmetric callose wall. Round and elongated mitochondria, plastids with internal membranes, small vacuoles and rough endoplasmic reticulum were observed in the dense cytoplasm of the microspores (Fig. 3b). Pollen wall formation was observed to be initiated at the tetrad stage by the formation of the exine template, called the primexine. At the early tetrad stage, the plasmalemma, which surrounded each microspore in a tetrad, was flat and in close contact with the callose wall enclosing the microspore (Fig. 3b). However, it later drew away from the callose wall
the plasmodesmata between the tapetal cell and sporogenous cell (SC sporogenous cell, N nucleus, Nu nucleolus, V vacuole, P plastid, M mitochondrion, Ep epidermis, En endothecium, ML middle layer, T tapetum)
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Fig. 2 TEM micrographs of longitudinal sections of spikelets at microspore mother cell stage. a Ultrastructure of microspore mother cells before meiosis. Note the numerous plastids and mitochondria in the cytoplasm of microspore mother cells. Note the presence of plasmodesmatal connections between adjacent cells (arrows). b
Fig. 3 TEM micrographs of microspores at tetrad stage of microsporogenesis. a General view of the tetrad in isobilateral arrangement. b Ultrastructure of microspore cytoplasm in a tetrad surrounded by callose wall. Note the plasmalemma is flat and in contact with the callose wall. Arrows show the presence of plastids with internal membranes. c Ultrastructure of a single microspore at late tetrad stage. Note the undulations of plasma membrane (white arrow) and deposition of primexine between plasmalemma and callose. The inset shows the formation of protectum and procolumellae (black arrow) (N nucleus, Nu nucleolus, Ca callose wall, V vacuole, P plastid, M mitochondrion, Pm plasma membrane, Pr primexine)
Ultrastructure of anther walls at microspore mother cell stage. Note binucleated tapetal cells. Arrows show the plasmodesmata between adjacent tapetal cells (N nucleus, Nu nucleolus, V vacuole, P plastid, M mitochondrion, Ep epidermis, En endothecium, ML middle layer, T tapetum)
Microsporogenesis and microgametogenesis in B. distachyon
and appeared undulated, and its surface became irregular (Fig. 3c). Degradation of the callose wall was also observed at this stage of development. The plasmalemma pulled away from the callose, and an electron-translucent primexine formed between the callose and the microspore plasmalemma (Fig. 3c). This plasmalemma surface coat (the primexine matrix) had a loose, irregular fibrillar texture. Some deposition of electron-dense material at a few sites within the primexine wall was observed as vertical elements, which appeared to be the beginnings of the protectum and procolumellae (Fig. 3c). Free microspore stage Following the dissolution of the callose envelope that holds the four microspores together at the tetrad stage, the microspores became free in the anther locule. The newly scattered mononuclear microspores were small, wrinkled and somewhat angular in shape (Fig. 4a). The microspores had large nuclei, prominent nucleoli, a vacuole-less cytoplasm and visibly thickened walls. With further development, the microspore nucleus migrated to the periphery and became flattened (referred to as offset, Fig. 4b). Few mitochondria were observed surrounding the microspore nucleus at this stage Fig. 4 Ultrastructure of microspores at free microspore stage of development. a General view of the microspore released from the callose wall. Note the irregular shape of the microspore. b Details of the microspore cytoplasm showing offset nucleus. Note the nucleus is surrounded by numerous mitochondria. Black arrows show the supratectal elements in exine. c Details of pollen wall formation and microspore cytoplasm. Formation of supratectal elements is shown by black arrows. Note the presence of microchannels in the tectum (white arrows). d, e Details of pore development. White arrows show the annulus (N nucleus, Nu nucleolus, M mitochondrion, Ekt ektexine, End endexine, Co columella, Te tectum, FL foot layer, Ub ubisch body, T tapetum)
(Fig. 4b). Exine construction, which began at the tetrad stage, progressed through the free microspore stage by the addition of sporopollenin from the tapetum to primexine. All the major components of the exine wall were visible at this stage. Both the ektexine and endexine were formed and can be easily distinguished in the TEM micrographs (Fig. 4b, c). The ektexine involves the formation of the basal foot layer, columella (baculum), thick tectum and supratectal elements. The presence of microchannels in the tectum was a general characteristic of pollen wall development (Fig. 4c). The rudimentary endexine was deposited as a thin, discontinuous layer between the foot layer and the plasma membrane. The pore sites were visible during the early free microspore stage (Fig. 4d), and observed as thickened aperture margin formed internal to the exine, that is called as endoaperture. The development of the pore was characterised by the thickening of the endexine, pressing outward by cytoplasmic expansions that resulted in a bulging ektexine ring known as annulus (Fig. 4e). Thus, the annulus is a product of a layer within the exine that is developed sufficiently to Braise^ the ektexine or cytoplasmic expansion sufficiently to press the layer within the exine outward. At this stage of development, the thickened components of the annulus formed a ring-shaped bulge and pressed outward (Fig. 4e). Ubisch bodies/Orbicules were also
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observed during this stage lining the inner tapetal wall (Fig. 4e). The Orbicules appeared spherical in shape with a hollow transparent central core, surrounded by a thin wall (Fig. 4e).
Vacuolated microspore stage
compressed except in the apertural region. Furthermore, the first deposit of the intine was observed as a thin, darkly stained, fibrillar layer called the Z-layer (Fig. 5e). During the formation of the intine, some Golgi-derived vesicles were deposited near the plasmalemma, thus contributing to Z-layer formation (Fig. 5e). By this stage of development, the Ubisch bodies were fully formed and seen deposited along the degenerated tapetal cells (Fig. 5f).
The microspores expanded in volume and became spherical with the formation of the large vacuole (Fig. 5a). A high degree of cytoplasmic activity was observed, with numerous mitochondria, Golgi vesicles and dictyosomes in the narrow strip of cytoplasm around the nucleus and the periphery of the cell (Fig. 5b). As reported previously (Sharma et al. 2014a), the orientation of the microspore pore was observed towards the tapetum (Fig. 5c). In the vacuolated microspore stage, the annulus was observed elevated. An operculum was also observed covering the pore (Fig. 5c). The exine layer of microspore had the same electron density as the Ubisch bodies (Fig. 5c, d). Ubisch bodies developed osmiophilic spikes (Fig. 5d). The foot layer thickened, and the endexine became
In the two-celled pollen grain, which was produced by asymmetric mitotic division, a lens-shaped generative cell was located adjacent to the vegetative cell (Fig. 6a). The beginning of the accumulation of reserve substances (starch granules, lipid bodies and granular particles) was observed to be correlated to the diminishing vacuole, as observed in the bicellular pollen grain cytoplasm. The generative cell was completely enclosed by the cytoplasm of the vegetative nucleus. The former possessed a nucleus, a conspicuous nucleolus and a
Fig. 5 TEM micrographs of vacuolated microspores. a Polar view of spherical microspore. Note the formation of big vacuole adjacent to the nucleus and a single pore. b Polar view of microspore nucleus surrounded by numerous mitochondria and dictyosomes. Black arrows show the presence of vesicles. c Vacuolated microspore with pore orientation towards the tapetum. Note the deposition of Ubisch bodies lining the tapetal wall. Black arrows show the raised annulus. d Details of Ubisch bodies lining the tapetal wall. Black arrow shows osmiophilic spikes in
Ubisch bodies. e Details of the pollen wall. The deposition of darkly stained, fibrillar Z-layer is shown by white arrows. Note the presence of Golgi-derived vesicles (black arrows) in the microspore cytoplasm. f Ultrastructure of anther walls at vacuolated stage. Note the degenerating tapetum and deposition of Ubisch bodies (N nucleus, Nu nucleolus, V vacuole, M mitochondrion, d dictyosome, Ub Ubisch body, Te tectum, O operculum, Mi microspore, FL foot layer, Ep epidermis, En endothecium, T tapetum)
Bicellular pollen stage
Microsporogenesis and microgametogenesis in B. distachyon
Fig. 6 TEM micrographs of bicellular pollen grain. a Polar view of twocelled pollen grain. Note the presence of lens-shaped generative cell, diminishing vacuole and beginning of starch accumulation. b Magnified view of lens-shaped generative cell showing reduced cytoplasm with only a few mitochondria. c Magnified view of vegetative nucleus surrounded by numerous mitochondria, starch grains and granular particles. d Magnified view of pollen cytoplasm showing starch grains, granular particles and mitochondria (black arrows). e Details of pore in bicellular pollen grain. Note the outward bulging of
aperture (asterisk). f Details of pollen wall. Note the presence of well developed exine with tectal microchannels (white arrows). Intine wall formation is in progress. Convolutions of Z-layer are shown by black arrow. g Ultrastructure of anther walls at bicellular pollen stage. Note the degraded tapetal cells and Ubisch bodies (VN vegetative nucleus, Nu nucleolus, V vacuole, GC generative cell, GN generative nucleus, S starch granule, M mitochondrion, Ex exine, In intine, Ub Ubisch body, Ep epidermis, En endothecium, T tapetum)
reduced cytoplasm with scarce mitochondria (Fig. 6b). The vegetative cell cytoplasm was dense, with abundant mitochondria; most of them were grouped at the periphery of the cell, though a few were observed lining the vegetative cell nucleus (Fig. 6c). With maturation, the starch grains and granular particles completely filled the cytoplasm of the bicellular pollen grain (Fig. 6d). At this stage of development, the apertural zone was observed bulging outward (Fig. 6e). Exine formation was completed by this stage, and numerous microchannels were still observed in the tectum (Fig. 6f). However, intine construction continued by the addition of materials from the vesicles produced by the microspore. The intine layer was observed to consist of fibrillar material that is slightly dense at its proximal surface where it is in direct contact with the plasma membrane. Both the exine and intine were well distinguishable during this stage. The Z-layer was still observed as thin, darkly stained and convoluted at some sites (Fig. 6f). As the intine increased in thickness, the endexine became compressed and, as a result, was no longer
discernible. The tapetal cells were compressed and seen degenerating, and Ubisch bodies were observed on the tapetal membrane next to the tangential wall of the endothecial cells (Fig. 6g). Mature pollen stage The mature pollen grain was formed by mitotic division of generative cell into two sperm cells. The pollen at this stage is oval in shape and trinucleate containing a vegetative nucleus and two sperm cells. The cytoplasm of the mature pollen grains appeared to be filled with ribosomes, starch granules, lipid bodies and granular particles (Fig. 7a, b). The plasmalemma became highly undulated at this stage and showed the deposition of a thin fibrillar layer that completed the formation of the intine (Fig. 7c). The Z-layer appeared more compact at this stage (Fig. 7c, d). The tectal microchannels were few compared to the bicellular stage (Fig. 7c). At this stage, the exine with conspicuous supratectum spines and a three-
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Fig. 7 TEM micrographs of mature pollen grain. a Polar view of mature pollen grain. Note the accumulation of reserve substances in the pollen cytoplasm. b Details of pollen cytoplasm showing ribosomes and starch granules. c Details of pollen wall. Note the undulations of plasma membrane and formation of well-developed exine and intine walls. Tectal microchannels are shown by white arrow. d Magnified view of
three-layered intine wall. The three layers are shown with black arrows. e Details of pollen pore. Note the raised annulus. f Ultrastructure of anther walls at mature pollen stage. Note the completely degraded tapetum with only remnants of Ubisch bodies (Ex exine, In intine, S starch granule, Ub ubisch body, Ep epidermis, En endothecium)
layered intine with numerous invaginations coated by the plasmalemma can be observed in the electron micrographs (Fig. 7c, d). The pore margin was observed compressed at this stage, but the annulus around the pore was raised (Fig. 7e). The operculum was no longer visible at this stage (Fig. 7e). The tapetal cells completely disappeared at the mature pollen stage of development and only remnants of Ubisch bodies/ Orbicules were observed lining the endothecial cells (Fig. 7f).
1989). This is a critical period during pollen development because the sporogenous cells prepare for meiosis. The nucleus might contribute considerable amounts of substances to the cytoplasm as a result of such evaginations and blebbing off of vesicles, in preparation for meiosis. In the next stage, microspore mother cells were observed connected to each other by cytoplasmic channels. According to Heslop-Harrison (1964, 1966), these connections allow the synchronic development of the archesporium. During this stage, the cytoplasm of microspore mother cells showed high metabolic activity with numerous elongated mitochondria, plastids, dictyosomes and vacuoles (Fig. 2a), suggesting their possible role in the production of callose. Following meiotic division, isobilateral tetrads are formed by successive cytokinesis. After release from the tetrad, free microspores begin to develop, undergo vacuolisation and mitotic divisions, leading to the production of mature bicellular and tricellular pollen grains. Male gametophyte development is of critical importance in sexual reproduction of flowering plants. Defects in pollen development can occur due to environmental factors such as heat stress that may lead to male sterility (Sakata et al. 2000; Prasad
Discussion Microsporogenesis and microgametogenesis in B. distachyon conform to the typical framework of grass pollen development (Harsant et al. 2013, Sharma et al. 2014a, b). An interesting activity of the nuclear membrane was observed during the sporogenous cell stage in B. distachyon that involves evaginations and blebbing off of the membrane of the nuclear envelope. Such activity indicates significant nucleo-cytoplasmic transfer or exchange. A similar activity of the nuclear membrane was observed in close relative, wheat (Mizelle et al.
Microsporogenesis and microgametogenesis in B. distachyon
et al. 2006; Endo et al. 2009; Farooq et al. 2011). A recent elaborative report on high temperature stress effects on pollen development in B. distachyon revealed pollen sterility due to arrested pollen development at vacuolated stage and abnormal tapetal development with enlarged and ruptured tapetal cells (Harsant et al. 2013). The tapetum in B. distachyon is of secretory type and degenerates during the course of pollen development. In the anthers of flowering plants with the secretory type of tapetum, bodies with chemical properties similar to those of the sporopollenin of pollen exine accumulate on the inner wall of the tapetal cells during pollen development. These bodies are known as Orbicules/Ubisch bodies. The detailed ontogeny of anther wall development and tapetal characteristics has been described in our previous study (Sharma et al. 2014b) where we observed that the Orbicules/Ubisch bodies originate from the tapetum and are involved in exine synthesis. The tapetum-specific expression of genes required for sporopollenin synthesis indicates that an abundance of sporopollenin is exported from tapetal cells (Ariizumi and Toriyama 2011; Liu and Fan 2013). Recently, immunolocalization and protein-protein interaction studies have supported a model in which the enzymes producing sporopollenin precursors are organized in a metabolon within the tapetum (Lallemand et al. 2013). This study clearly shows that the aperture in B. distachyon is a highly specialized region of pollen grain wall, which undergo considerable structural changes during pollen development. Although early studies on grass pollen development have reported that the aperture (pore) sites become visible at the late tetrad stage of pollen development (Rowley, 1962, 1964; Heslop-Harrison 1963), we could observe these pore sites only at the free microspore stage in B. distachyon. The apertural region is one of the most significant features of the pollen grain and has been widely used for taxonomic purposes. This aperture which serves both as a harmomegathy and a germinal exit is a characteristic of many monocots. B. distachyon shows the archetypal aperture system which is typical in grasses. The archetypal aperture system in grass pollen is a single pore consisting of an Bisland^ of ectoaperture exine, the operculum (Wodehouse 1935), circumscribed by a raised and thickened border of ektexine, the annulus (Jackson 1928; Punt et al. 1994). Proximally (i.e. facing the cytoplasm), a thickened ring-like layer (thickening of the endexine), surrounds the pore margin. At the free microspore stage and in subsequent stages, the apertural zone is distinguishable from the rest of the wall by its thickness, shape and substructure. As the pollen grain grows, the apertural zone increases in diameter. In the early stages of microspore development in B. distachyon, the lamellae that make up the thickened aperture margin formed internal to the exine around what could be called an endoaperture (Fig. 4d). Similar observations were made in Poa annua (Rowley 1964), Zea mays (Skvarla and Larson 1966) and Triticum aestivum (El-
Ghazaly and Jensen 1987, 1990). At late free microspore stage and vacuolated stage of development, the pore margins were seen elevated forming a raised annulus (Figs. 4e and 5c). However, raised annulus was observed later in mature stages of pollen development in Z. mays (Skvarla and Larson 1966) and Sorghum bicolor (Christensen and Horner 1974). During vacuolated microspore stage, the endexine becomes compressed except in the apertural region (Fig. 5c). The highly developed endexine in the annulus probably protects the aperture margin from collapsing during vacuolation of microspores. In the mature pollen stage, the pore margin became compressed that it no longer protrude internally and the annulus around the pore has the raised aspect typical of most pollen grains of Poaceae (Skvarla and Larson 1966; Christensen and Horner 1974; El-Ghazaly and Jensen 1987). Ontogeny of the pollen wall This is the first study that delineates pollen wall developmental stages in B. distachyon. Published observations show that the pollen wall template is determined during meiosis or just after meiosis (Heslop-Harrison 1963; Sheldon and Dickinson 1983, 1986; Takahashi 1989, 1994; Zhou et al. 2015). However, different perspectives on exine pattern formation have been proposed. Rowley and Skvarla (1975), Rowley and Dahl (1977) and Rowley (1990) asserted that a plasma membrane-associated compound, glycocalyx, determines the initial exine pattern. According to Heslop-Harrison (1963); Zhou et al. 2015, the exine pattern is determined by the primexine and begins with the formation of probacules in the primexine matrix. However, Takahashi (1989, 1993, 1994) and Takahashi and Skvarla (1991a, b) proposed that the plasma membrane rather than the primexine is responsible for exine pattern determination. According to these authors, the exine pattern is determined by invagination of the microspore plasma membrane at the early tetrad stage, and the protectum is formed directly on the plasma membrane before primexine matrix and probacule formation (Takahashi and Kouchi 1988; Takahashi 1989). Skvarla and Larson (1966) had previously described the primexine as the ‘exine template’ because of the organized manner in which it accumulates sporopollenin at specific sites. Primexine is a microfibrillar material composed largely of cellulose and functions as an elaborate glycocalyx in which the patterned accumulation of sporopollenin precursors and their subsequent polymerization occurs (Rowley and Dahl 1977). In the present study, exine formation in B. distachyon was found to initiate with the undulation of plasma membrane at the late tetrad stage and was followed by primexine formation. The small vesicles and vacuoles in close contact with the plasmalemma (Fig. 4) at the tetrad stage indicate that materials deposited at the special sites of the primexine are produced by the microspore. At the free microspore stage, the exine was
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well defined, with the formation of the ektexine (tectum, supratectal elements, columellae and foot layer) and endexine. The tectum was observed dissected with a few microchannels (Fig. 6a). In wheat, the tectum is observed as discontinuous and interrupted with radially oriented microchannels (ElGhazaly and Jensen 1986). However, microchannels were not identified in the foot layer of B. distachyon in the present study. The presence of more microchannels in the tectum than in the foot layer is a general observation for members of Poaceae (Skvarla and Larson 1966; Christensen et al. 1972; Heslop-Harrison 1979; Marquez et al. 1997b). In rice and maize, the presence of numerous microchannels, in both the tectum and the foot layer, has been reported; however, in the same study, the pennisetum pollen wall showed the absence of microchannels in the tectum and the foot layer (Fu et al. 2001). Many authors (Rowley 1973; El-Ghazaly and Jensen 1985) have considered these tectal microchannels to be adaptive mechanisms that help in the interchanging of materials from the inside to the outside of the pollen grain. However, Lin et al. (1979) believed that microchannels are related to the release of proteins and pollen grain recognition by the stigmas. The formation of the tectum begins during the tetrad stage of pollen development in Triticum (El-Ghazaly and Jensen 1986), Lilium (Heslop-Harrison 1971) and Zea (Skvarla and Larson 1966). In Sorghum (Christensen et al. 1972), however, the tectum appears to develop only after the microspores are released from the callose wall. A similar ontogenetic timing for tectum development was observed in B. distachyon during the free microspore stage of development. The endexine develops as a rudimentary, thin layer on the proximal surface of the foot layer during the free microspore stage of development. In B. distachyon, the intine is the last layer to be laid down. In the next stage, during vacuolation, the endexine is pressed against the proximal surface of the foot layer after the formation of the Z-layer, making it difficult to observe. The peripheral cytoplasm at the vacuolate stage of development is dense with Golgi vesicles and dictyosomes, and the vesicles fuse with the plasmalemma, possibly contributing to the formation of the intine (Fernandez and Rodriguez-Garcia 1989). As observed in earlier studies, the distal face of the intine is formed by material transported in Golgi vesicles (Echlin and Godwin 1969; Roland 1971). Z-layer formation is the first contribution to the formation of the intine, though the well developed intine wall is observed after mitosis, during the bicellular pollen stage. A similar timing of intine development has been reported for Triticum (El-Ghazaly and Jensen 1986), Zea (Skvarla and Larson 1966) and Sorghum (Christensen et al. 1972). However, in Lilium, Silene (Heslop-Harrison 1963, 1968a) and Arabidopsis (Owen and Makaroff 1995), the intine develops before mitosis during the free microspore stage of development. Another characteristic feature in the development of the intine is the number of strata it contains. In Poaceae
(Marquez et al. 1997a, b) and Euphorbiaceae (Suarez-Cervera et al. 2001), three layers of intine have been reported. However, in some species, such as Nelumbo lutea (Kreunen and Osborn 1999), Hypecoum imberbe (Romero et al. 2003) and Tarenna gracilipes (Vinckier and Smets 2005), two strata have been reported. Furthermore, a single homogenous stratum has been described in Olea europaea (Fernandez and Rodriguez-Garcia 1988) and Lycopersicon esculentum (Fernandez et al. 1992). In the present study, TEM observations of the mature pollen wall of B. distachyon revealed the presence of a three-layered intine wall. In the current study, we have described the pollen ontogenic sequence in B. distachyon, including the ultrastructure of the mature pollen wall. In particular, the pattern of microsporogenesis, exine formation and aperture development were evaluated for the first time in this model plant. Developmental timing of exine components and tectal microchannels supports the hypothesis that these elements of the exine function in pollenkitt storage. Moreover, absence of microchannels in the foot layer of B. distachyon also illuminates ontogenic differences between B. distachyon and its close member wheat, which reflect reproductive adaptations with evolution. Acknowledgments We thank Dr. Simon Crawford for technical guidance with electron microscopy and access to Advanced Microscopy Facility, The University of Melbourne. AS also thanks Dr. Martin O’Brien for helping in identifying the stages of anther and pollen development and Dr. Lim Chee Liew for suggesting improvements in the manuscript and helping in organizing the figures during manuscript preparation. Financial support from the Australian Research Council (ARC DPO988972) is also gratefully acknowledged. Conflict of interest The authors declare that they have no conflict of interest.
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ORIGINAL ARTICLE
Ultrastructure of microsporogenesis and microgametogenesis in Brachypodium distachyon Akanksha Sharma & Mohan B. Singh & Prem L. Bhalla
Received: 16 October 2014 / Accepted: 2 March 2015 # Springer-Verlag Wien 2015
Abstract Brachypodium distachyon has emerged as a model system for forage grass and cereal grain species. Here, we report B. distachyon pollen development at the ultrastructural level. The process of microsporogenesis and microgametogenesis in B. distachyon follows the typical angiosperm pollen development sequence. Pronounced evaginations of the nuclear envelope are observed prior to meiosis, indicating active nucleocytoplasmic exchange processes. The microspore mother cells undergo meiosis and subsequent cytokinesis, forming isobilateral tetrads. Following dissolution of the callose wall and release of free and vacuolated microspores, mitotic divisions lead to the formation of mature, three-celled pollen grains. In B. distachyon, pollen wall formation begins at the tetrad stage by the formation of the exine template (primexine). The exine is tectate-columellate, comprising a foot layer and endexine. Development of the tectum and the foot layer is complete by the free microspore stage of development, with the tectum formed discontinuously. The endexine initiates in the free microspore stage but becomes compressed in mature grains. The intine layer is deposited after mitosis and comprises three layers during the mature pollen stage of development. Pore development initiates during early free microspore development stage and Brachypodium pollen has a single germination pore consisting of a slightly raised annulus surrounding a central operculum. The tapetum is of the secretory type with loss of the tapetal cell walls beginning at about the time of microsporocyte meiosis. This is the first report on ultrastructure of microsporogenesis and microgametogenesis in B. distachyon. In general,
Handling Editor: Benedikt Kost A. Sharma : M. B. Singh : P. L. Bhalla (*) Plant Molecular Biology and Biotechnology Laboratory, Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, Melbourne, VIC 3010, Australia e-mail: [email protected]
Brachypodium microsporogenesis and microgametogenesis conform to a typical grass pollen development pattern. Keywords Brachypodium distachyon . Ontogeny . Pollen . Pollen wall . Ultrastructure . Microsporogenesis . Microgametogenesis
Introduction Male reproductive development in flowering plants is initiated by the formation of stamens that house sporogenous cells, the microsporocytes. Microsporogenesis comprises the sequence of successive developmental stages from sporogenous cells to haploid unicellular microspores. During this process, the microsporocytes undergo meiotic division to generate four haploid microspores (tetrad) enclosed within a callose wall. Microgametogenesis begins at the release of haploid microspores from tetrads. The haploid microspores then undergo asymmetric division to give rise to bicellular pollen that contains a smaller generative cell enclosed within a larger vegetative cell. The generative cell further divides to give rise to the two sperm cells required for double fertilization. This study focuses on the ultrastructural changes occurring throughout the process of microsporogenesis and microgametogenesis in Brachypodium distachyon. B. distachyon, a member of the Pooideae subfamily, is a small annual grass with biological, physical and genomic attributes of a model plant for grasses and cereal crops (Draper et al. 2001; Garvin et al. 2008; Opanowicz et al. 2008; Bevan et al. 2010; Girin et al. 2014). It is endemic to the Mediterranean and Middle East and belongs to the Bcore pooid^ genera that include the majority of important temperate cereals and forage grasses, such as wheat, barley and switch grass (Draper et al. 2001).
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Deciphering the morphological events involved in microsporogenesis and microsporogenesis is critical for understanding the reproductive biology and phylogeny of flowering plants (Blackmore et al. 2007). The study of pollen development for the genus Brachypodium has revealed that it has trinucleate, uniporate, spheroidal pollen grains with granular ornamentation. The microspore cytoplasm contains variable amounts of insoluble polysaccharides and proteins at different developmental stages. Anther wall development follows the monocotyledonous-type, which is composed of an epidermal layer, an endothecial layer, one middle layer and a secretorytype tapetum (Harsant et al. 2013; Sharma et al. 2014a, b). The ultrastructural features of pollen development have been addressed in other members of the Pooideae family, such as wheat (El-Ghazaly and Jensen 1986; Mizelle et al. 1989), maize (Skvarla and Larson 1966) and sorghum (Christensen et al. 1972; Christensen and Horner 1974). In contrast, there is no study available to date on the ultrastructural features of pollen development in the genus Brachypodium. The present study integrates ultrastructural and developmental observations to document critical pollen developmental events in B. distachyon using transmission electron microscopy (TEM). We paid special attention to B. distachyon pollen wall development, as there are significant variations in the pollen wall architecture and timing of distinct developmental stages between species.
Materials and methods Fresh immature spikelets and anthers at various stages of development were collected from B. distachyon (Bd21-3) plants on different days based on the visibility of the first awn out of the flag leaf to the stage when dehisced anthers were noticeable (a period of 9 days). Spikelets and anthers were classified into groups according to the related stage of pollen development (Table 1). Their stage of development had been previously determined by examining LR White-embedded sections using light microscopy (Sharma et al. 2014a). For the transmission electron microscopy (TEM) studies, spikelets and anthers were fixed in cold 2.5 % glutaraldehyde/ 0.1 M PBS (phosphate-buffered saline), pH 7.0, for 4 h at room temperature. Following dehydration in an ethanol series, the material was then incubated in 1:1 and 1:3 ethanol:resin (LR White) mixtures for 1 day each at room temperature and finally overnight in 100 % resin on a rotating platform. Following dehydration, the spikelets and anthers were embedded in LR White, which was polymerized by incubation at 60 °C for 24–30 h. Ultrathin sections (90 nm) were cut using a Leica Ultracut® microtome and then stained with 2 % uranyl acetate for 10 min, followed by triple lead stain for 5 min (Sato 1968). The sections were observed and photographed with a Philips CM120 BioTwin transmission electron microscope.
Terminology The terminology used to describe the different exine layers of B. distachyon is defined according to Hesse et al. (2009).
Results Although microsporogenesis and microgametogenesis comprise a continuous process, to clearly describe the events occurring during pollen ontogeny in B. distachyon, the process is classified into seven developmental stages: sporogenous cell, microspore mother cell, tetrad, free microspore, vacuolated microspore, bicellular pollen and mature pollen stages. Sporogenous cell stage At this stage, the microsporocytes or the sporogenous cells were slightly polygonal and tightly packed to completely fill the locular space. The sporogenous cells were usually arranged into two rows on longitudinal sections. These cells contained large, centrally located nuclei with prominent nucleoli, exhibited thin walls and were connected to each other by plasmodesmata (Fig. 1a). A few vacuoles, plastids and mitochondria were observed in the cytoplasm of the sporogenous cells (Fig. 1a). The nuclei of these cells showed scattered and diffuse chromatin. The outer membrane of the nuclear envelope was observed to be evaginated at a few sites (Fig. 1a), possibly constituting a line of transport and exchange between the nucleus and cytoplasm. During the sporogenous cell stage, the anther wall completed its differentiation, comprising of four layers: the epidermis, the endothecium, the middle layer and the tapetum (Fig. 1b). Plasmodesmatal connections were observed between adjacent tapetal cells and between the microsporocytes and tapetal cells (Fig. 1b). The tapetal cells were rectangular in outline and possessed a single large nucleus. Their cytoplasm stained more densely, in contrast with the microspore cytoplasm. Small vacuoles, few plastids and mitochondria were observed in tapetal cytoplasm (Fig. 1b). Microspore mother cell stage At the premeiotic stage, the microspore mother cells showed a centrally located, large nucleus and nucleolus and were connected to each other by cytoplasmic connections. At this stage, numerous plastids, mitochondria and slightly bigger vacuoles were observed throughout the microsporocyte cytoplasm (Fig. 2a). Many of the mitochondria were observed to be elongated and were most likely in the process of dividing. Few plastids of the microspore mother cells were observed elongated or containing internal membranes (Fig. 2a). Just before meiotic divisions, the microsporocytes became less
Microsporogenesis and microgametogenesis in B. distachyon Table 1
Temporal developmental stages of anther and microspores of B. distachyon (Sharma et al. 2014a)
Daysa
Plant material fixed and sectioned
No. of stages identified
Stages identified
Stage of the base floret
1 2
Spikelet Spikelet
2 3
Primary parietal cell stage Pollen mother cell stage
3
Spikelet
4
4
Spikelet
3
5
Spikelet
3
6 7 8 9
Anther Anther Anther Anther
1 1 1 1
Archesporial cell stage, primary parietal cell stage Primary sporogenous cell stage, sporogenous cell stage and pollen mother cell stage Primary parietal cell stage, primary sporogenous cell stage, sporogenous cell stage and Pollen mother cell stage Sporogenous cell stage, pollen mother cell stage and tetrad stage Pollen mother cell stage, tetrad stage and free microspore stage Vacuolated microspore stage Vacuolated microspore stage Bicellular pollen stage Mature pollen stage
Pollen mother cell stage
Tetrad stage Free microspore stage – – – –
a
Day 1: when the first awn was just visible out of the flag leaf; days 2–9: subsequent days after awn visibility; day 9: anthesis, when yellow, mature anthers were clearly visible
polygonal, elliptical or irregular, and were still connected to each other. The tapetal cells undergo nuclear division and appeared binucleated at this stage and were connected to each other and to the pollen mother cells by their original plasmodesmata (Fig. 2b). At this stage, tapetal cells have a greater density of cytoplasm than in the previous stage with numerous mitochondria, plastids and few small vacuoles (Fig. 2b). Tetrad stage After meiosis, tetrads of haploid microspores randomly filled the entire space of the anther locules. Following successive
Fig. 1 TEM micrographs of longitudinal sections of spikelets at sporogenous cell stage. a Ultrastructure of sporogenous cells. Black arrows show the plasmodesmata between adjacent sporogenous cells. White arrows show the evaginations of the nuclear membrane. b Ultrastructure of anther walls at sporogenous cell stage. Arrows show
cytokinesis, numerous tetrads with isobilateral arrangement (Fig. 3a) were observed. Each tetrad enclosed four haploid microspores within an asymmetric callose wall. Round and elongated mitochondria, plastids with internal membranes, small vacuoles and rough endoplasmic reticulum were observed in the dense cytoplasm of the microspores (Fig. 3b). Pollen wall formation was observed to be initiated at the tetrad stage by the formation of the exine template, called the primexine. At the early tetrad stage, the plasmalemma, which surrounded each microspore in a tetrad, was flat and in close contact with the callose wall enclosing the microspore (Fig. 3b). However, it later drew away from the callose wall
the plasmodesmata between the tapetal cell and sporogenous cell (SC sporogenous cell, N nucleus, Nu nucleolus, V vacuole, P plastid, M mitochondrion, Ep epidermis, En endothecium, ML middle layer, T tapetum)
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Fig. 2 TEM micrographs of longitudinal sections of spikelets at microspore mother cell stage. a Ultrastructure of microspore mother cells before meiosis. Note the numerous plastids and mitochondria in the cytoplasm of microspore mother cells. Note the presence of plasmodesmatal connections between adjacent cells (arrows). b
Fig. 3 TEM micrographs of microspores at tetrad stage of microsporogenesis. a General view of the tetrad in isobilateral arrangement. b Ultrastructure of microspore cytoplasm in a tetrad surrounded by callose wall. Note the plasmalemma is flat and in contact with the callose wall. Arrows show the presence of plastids with internal membranes. c Ultrastructure of a single microspore at late tetrad stage. Note the undulations of plasma membrane (white arrow) and deposition of primexine between plasmalemma and callose. The inset shows the formation of protectum and procolumellae (black arrow) (N nucleus, Nu nucleolus, Ca callose wall, V vacuole, P plastid, M mitochondrion, Pm plasma membrane, Pr primexine)
Ultrastructure of anther walls at microspore mother cell stage. Note binucleated tapetal cells. Arrows show the plasmodesmata between adjacent tapetal cells (N nucleus, Nu nucleolus, V vacuole, P plastid, M mitochondrion, Ep epidermis, En endothecium, ML middle layer, T tapetum)
Microsporogenesis and microgametogenesis in B. distachyon
and appeared undulated, and its surface became irregular (Fig. 3c). Degradation of the callose wall was also observed at this stage of development. The plasmalemma pulled away from the callose, and an electron-translucent primexine formed between the callose and the microspore plasmalemma (Fig. 3c). This plasmalemma surface coat (the primexine matrix) had a loose, irregular fibrillar texture. Some deposition of electron-dense material at a few sites within the primexine wall was observed as vertical elements, which appeared to be the beginnings of the protectum and procolumellae (Fig. 3c). Free microspore stage Following the dissolution of the callose envelope that holds the four microspores together at the tetrad stage, the microspores became free in the anther locule. The newly scattered mononuclear microspores were small, wrinkled and somewhat angular in shape (Fig. 4a). The microspores had large nuclei, prominent nucleoli, a vacuole-less cytoplasm and visibly thickened walls. With further development, the microspore nucleus migrated to the periphery and became flattened (referred to as offset, Fig. 4b). Few mitochondria were observed surrounding the microspore nucleus at this stage Fig. 4 Ultrastructure of microspores at free microspore stage of development. a General view of the microspore released from the callose wall. Note the irregular shape of the microspore. b Details of the microspore cytoplasm showing offset nucleus. Note the nucleus is surrounded by numerous mitochondria. Black arrows show the supratectal elements in exine. c Details of pollen wall formation and microspore cytoplasm. Formation of supratectal elements is shown by black arrows. Note the presence of microchannels in the tectum (white arrows). d, e Details of pore development. White arrows show the annulus (N nucleus, Nu nucleolus, M mitochondrion, Ekt ektexine, End endexine, Co columella, Te tectum, FL foot layer, Ub ubisch body, T tapetum)
(Fig. 4b). Exine construction, which began at the tetrad stage, progressed through the free microspore stage by the addition of sporopollenin from the tapetum to primexine. All the major components of the exine wall were visible at this stage. Both the ektexine and endexine were formed and can be easily distinguished in the TEM micrographs (Fig. 4b, c). The ektexine involves the formation of the basal foot layer, columella (baculum), thick tectum and supratectal elements. The presence of microchannels in the tectum was a general characteristic of pollen wall development (Fig. 4c). The rudimentary endexine was deposited as a thin, discontinuous layer between the foot layer and the plasma membrane. The pore sites were visible during the early free microspore stage (Fig. 4d), and observed as thickened aperture margin formed internal to the exine, that is called as endoaperture. The development of the pore was characterised by the thickening of the endexine, pressing outward by cytoplasmic expansions that resulted in a bulging ektexine ring known as annulus (Fig. 4e). Thus, the annulus is a product of a layer within the exine that is developed sufficiently to Braise^ the ektexine or cytoplasmic expansion sufficiently to press the layer within the exine outward. At this stage of development, the thickened components of the annulus formed a ring-shaped bulge and pressed outward (Fig. 4e). Ubisch bodies/Orbicules were also
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observed during this stage lining the inner tapetal wall (Fig. 4e). The Orbicules appeared spherical in shape with a hollow transparent central core, surrounded by a thin wall (Fig. 4e).
Vacuolated microspore stage
compressed except in the apertural region. Furthermore, the first deposit of the intine was observed as a thin, darkly stained, fibrillar layer called the Z-layer (Fig. 5e). During the formation of the intine, some Golgi-derived vesicles were deposited near the plasmalemma, thus contributing to Z-layer formation (Fig. 5e). By this stage of development, the Ubisch bodies were fully formed and seen deposited along the degenerated tapetal cells (Fig. 5f).
The microspores expanded in volume and became spherical with the formation of the large vacuole (Fig. 5a). A high degree of cytoplasmic activity was observed, with numerous mitochondria, Golgi vesicles and dictyosomes in the narrow strip of cytoplasm around the nucleus and the periphery of the cell (Fig. 5b). As reported previously (Sharma et al. 2014a), the orientation of the microspore pore was observed towards the tapetum (Fig. 5c). In the vacuolated microspore stage, the annulus was observed elevated. An operculum was also observed covering the pore (Fig. 5c). The exine layer of microspore had the same electron density as the Ubisch bodies (Fig. 5c, d). Ubisch bodies developed osmiophilic spikes (Fig. 5d). The foot layer thickened, and the endexine became
In the two-celled pollen grain, which was produced by asymmetric mitotic division, a lens-shaped generative cell was located adjacent to the vegetative cell (Fig. 6a). The beginning of the accumulation of reserve substances (starch granules, lipid bodies and granular particles) was observed to be correlated to the diminishing vacuole, as observed in the bicellular pollen grain cytoplasm. The generative cell was completely enclosed by the cytoplasm of the vegetative nucleus. The former possessed a nucleus, a conspicuous nucleolus and a
Fig. 5 TEM micrographs of vacuolated microspores. a Polar view of spherical microspore. Note the formation of big vacuole adjacent to the nucleus and a single pore. b Polar view of microspore nucleus surrounded by numerous mitochondria and dictyosomes. Black arrows show the presence of vesicles. c Vacuolated microspore with pore orientation towards the tapetum. Note the deposition of Ubisch bodies lining the tapetal wall. Black arrows show the raised annulus. d Details of Ubisch bodies lining the tapetal wall. Black arrow shows osmiophilic spikes in
Ubisch bodies. e Details of the pollen wall. The deposition of darkly stained, fibrillar Z-layer is shown by white arrows. Note the presence of Golgi-derived vesicles (black arrows) in the microspore cytoplasm. f Ultrastructure of anther walls at vacuolated stage. Note the degenerating tapetum and deposition of Ubisch bodies (N nucleus, Nu nucleolus, V vacuole, M mitochondrion, d dictyosome, Ub Ubisch body, Te tectum, O operculum, Mi microspore, FL foot layer, Ep epidermis, En endothecium, T tapetum)
Bicellular pollen stage
Microsporogenesis and microgametogenesis in B. distachyon
Fig. 6 TEM micrographs of bicellular pollen grain. a Polar view of twocelled pollen grain. Note the presence of lens-shaped generative cell, diminishing vacuole and beginning of starch accumulation. b Magnified view of lens-shaped generative cell showing reduced cytoplasm with only a few mitochondria. c Magnified view of vegetative nucleus surrounded by numerous mitochondria, starch grains and granular particles. d Magnified view of pollen cytoplasm showing starch grains, granular particles and mitochondria (black arrows). e Details of pore in bicellular pollen grain. Note the outward bulging of
aperture (asterisk). f Details of pollen wall. Note the presence of well developed exine with tectal microchannels (white arrows). Intine wall formation is in progress. Convolutions of Z-layer are shown by black arrow. g Ultrastructure of anther walls at bicellular pollen stage. Note the degraded tapetal cells and Ubisch bodies (VN vegetative nucleus, Nu nucleolus, V vacuole, GC generative cell, GN generative nucleus, S starch granule, M mitochondrion, Ex exine, In intine, Ub Ubisch body, Ep epidermis, En endothecium, T tapetum)
reduced cytoplasm with scarce mitochondria (Fig. 6b). The vegetative cell cytoplasm was dense, with abundant mitochondria; most of them were grouped at the periphery of the cell, though a few were observed lining the vegetative cell nucleus (Fig. 6c). With maturation, the starch grains and granular particles completely filled the cytoplasm of the bicellular pollen grain (Fig. 6d). At this stage of development, the apertural zone was observed bulging outward (Fig. 6e). Exine formation was completed by this stage, and numerous microchannels were still observed in the tectum (Fig. 6f). However, intine construction continued by the addition of materials from the vesicles produced by the microspore. The intine layer was observed to consist of fibrillar material that is slightly dense at its proximal surface where it is in direct contact with the plasma membrane. Both the exine and intine were well distinguishable during this stage. The Z-layer was still observed as thin, darkly stained and convoluted at some sites (Fig. 6f). As the intine increased in thickness, the endexine became compressed and, as a result, was no longer
discernible. The tapetal cells were compressed and seen degenerating, and Ubisch bodies were observed on the tapetal membrane next to the tangential wall of the endothecial cells (Fig. 6g). Mature pollen stage The mature pollen grain was formed by mitotic division of generative cell into two sperm cells. The pollen at this stage is oval in shape and trinucleate containing a vegetative nucleus and two sperm cells. The cytoplasm of the mature pollen grains appeared to be filled with ribosomes, starch granules, lipid bodies and granular particles (Fig. 7a, b). The plasmalemma became highly undulated at this stage and showed the deposition of a thin fibrillar layer that completed the formation of the intine (Fig. 7c). The Z-layer appeared more compact at this stage (Fig. 7c, d). The tectal microchannels were few compared to the bicellular stage (Fig. 7c). At this stage, the exine with conspicuous supratectum spines and a three-
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Fig. 7 TEM micrographs of mature pollen grain. a Polar view of mature pollen grain. Note the accumulation of reserve substances in the pollen cytoplasm. b Details of pollen cytoplasm showing ribosomes and starch granules. c Details of pollen wall. Note the undulations of plasma membrane and formation of well-developed exine and intine walls. Tectal microchannels are shown by white arrow. d Magnified view of
three-layered intine wall. The three layers are shown with black arrows. e Details of pollen pore. Note the raised annulus. f Ultrastructure of anther walls at mature pollen stage. Note the completely degraded tapetum with only remnants of Ubisch bodies (Ex exine, In intine, S starch granule, Ub ubisch body, Ep epidermis, En endothecium)
layered intine with numerous invaginations coated by the plasmalemma can be observed in the electron micrographs (Fig. 7c, d). The pore margin was observed compressed at this stage, but the annulus around the pore was raised (Fig. 7e). The operculum was no longer visible at this stage (Fig. 7e). The tapetal cells completely disappeared at the mature pollen stage of development and only remnants of Ubisch bodies/ Orbicules were observed lining the endothecial cells (Fig. 7f).
1989). This is a critical period during pollen development because the sporogenous cells prepare for meiosis. The nucleus might contribute considerable amounts of substances to the cytoplasm as a result of such evaginations and blebbing off of vesicles, in preparation for meiosis. In the next stage, microspore mother cells were observed connected to each other by cytoplasmic channels. According to Heslop-Harrison (1964, 1966), these connections allow the synchronic development of the archesporium. During this stage, the cytoplasm of microspore mother cells showed high metabolic activity with numerous elongated mitochondria, plastids, dictyosomes and vacuoles (Fig. 2a), suggesting their possible role in the production of callose. Following meiotic division, isobilateral tetrads are formed by successive cytokinesis. After release from the tetrad, free microspores begin to develop, undergo vacuolisation and mitotic divisions, leading to the production of mature bicellular and tricellular pollen grains. Male gametophyte development is of critical importance in sexual reproduction of flowering plants. Defects in pollen development can occur due to environmental factors such as heat stress that may lead to male sterility (Sakata et al. 2000; Prasad
Discussion Microsporogenesis and microgametogenesis in B. distachyon conform to the typical framework of grass pollen development (Harsant et al. 2013, Sharma et al. 2014a, b). An interesting activity of the nuclear membrane was observed during the sporogenous cell stage in B. distachyon that involves evaginations and blebbing off of the membrane of the nuclear envelope. Such activity indicates significant nucleo-cytoplasmic transfer or exchange. A similar activity of the nuclear membrane was observed in close relative, wheat (Mizelle et al.
Microsporogenesis and microgametogenesis in B. distachyon
et al. 2006; Endo et al. 2009; Farooq et al. 2011). A recent elaborative report on high temperature stress effects on pollen development in B. distachyon revealed pollen sterility due to arrested pollen development at vacuolated stage and abnormal tapetal development with enlarged and ruptured tapetal cells (Harsant et al. 2013). The tapetum in B. distachyon is of secretory type and degenerates during the course of pollen development. In the anthers of flowering plants with the secretory type of tapetum, bodies with chemical properties similar to those of the sporopollenin of pollen exine accumulate on the inner wall of the tapetal cells during pollen development. These bodies are known as Orbicules/Ubisch bodies. The detailed ontogeny of anther wall development and tapetal characteristics has been described in our previous study (Sharma et al. 2014b) where we observed that the Orbicules/Ubisch bodies originate from the tapetum and are involved in exine synthesis. The tapetum-specific expression of genes required for sporopollenin synthesis indicates that an abundance of sporopollenin is exported from tapetal cells (Ariizumi and Toriyama 2011; Liu and Fan 2013). Recently, immunolocalization and protein-protein interaction studies have supported a model in which the enzymes producing sporopollenin precursors are organized in a metabolon within the tapetum (Lallemand et al. 2013). This study clearly shows that the aperture in B. distachyon is a highly specialized region of pollen grain wall, which undergo considerable structural changes during pollen development. Although early studies on grass pollen development have reported that the aperture (pore) sites become visible at the late tetrad stage of pollen development (Rowley, 1962, 1964; Heslop-Harrison 1963), we could observe these pore sites only at the free microspore stage in B. distachyon. The apertural region is one of the most significant features of the pollen grain and has been widely used for taxonomic purposes. This aperture which serves both as a harmomegathy and a germinal exit is a characteristic of many monocots. B. distachyon shows the archetypal aperture system which is typical in grasses. The archetypal aperture system in grass pollen is a single pore consisting of an Bisland^ of ectoaperture exine, the operculum (Wodehouse 1935), circumscribed by a raised and thickened border of ektexine, the annulus (Jackson 1928; Punt et al. 1994). Proximally (i.e. facing the cytoplasm), a thickened ring-like layer (thickening of the endexine), surrounds the pore margin. At the free microspore stage and in subsequent stages, the apertural zone is distinguishable from the rest of the wall by its thickness, shape and substructure. As the pollen grain grows, the apertural zone increases in diameter. In the early stages of microspore development in B. distachyon, the lamellae that make up the thickened aperture margin formed internal to the exine around what could be called an endoaperture (Fig. 4d). Similar observations were made in Poa annua (Rowley 1964), Zea mays (Skvarla and Larson 1966) and Triticum aestivum (El-
Ghazaly and Jensen 1987, 1990). At late free microspore stage and vacuolated stage of development, the pore margins were seen elevated forming a raised annulus (Figs. 4e and 5c). However, raised annulus was observed later in mature stages of pollen development in Z. mays (Skvarla and Larson 1966) and Sorghum bicolor (Christensen and Horner 1974). During vacuolated microspore stage, the endexine becomes compressed except in the apertural region (Fig. 5c). The highly developed endexine in the annulus probably protects the aperture margin from collapsing during vacuolation of microspores. In the mature pollen stage, the pore margin became compressed that it no longer protrude internally and the annulus around the pore has the raised aspect typical of most pollen grains of Poaceae (Skvarla and Larson 1966; Christensen and Horner 1974; El-Ghazaly and Jensen 1987). Ontogeny of the pollen wall This is the first study that delineates pollen wall developmental stages in B. distachyon. Published observations show that the pollen wall template is determined during meiosis or just after meiosis (Heslop-Harrison 1963; Sheldon and Dickinson 1983, 1986; Takahashi 1989, 1994; Zhou et al. 2015). However, different perspectives on exine pattern formation have been proposed. Rowley and Skvarla (1975), Rowley and Dahl (1977) and Rowley (1990) asserted that a plasma membrane-associated compound, glycocalyx, determines the initial exine pattern. According to Heslop-Harrison (1963); Zhou et al. 2015, the exine pattern is determined by the primexine and begins with the formation of probacules in the primexine matrix. However, Takahashi (1989, 1993, 1994) and Takahashi and Skvarla (1991a, b) proposed that the plasma membrane rather than the primexine is responsible for exine pattern determination. According to these authors, the exine pattern is determined by invagination of the microspore plasma membrane at the early tetrad stage, and the protectum is formed directly on the plasma membrane before primexine matrix and probacule formation (Takahashi and Kouchi 1988; Takahashi 1989). Skvarla and Larson (1966) had previously described the primexine as the ‘exine template’ because of the organized manner in which it accumulates sporopollenin at specific sites. Primexine is a microfibrillar material composed largely of cellulose and functions as an elaborate glycocalyx in which the patterned accumulation of sporopollenin precursors and their subsequent polymerization occurs (Rowley and Dahl 1977). In the present study, exine formation in B. distachyon was found to initiate with the undulation of plasma membrane at the late tetrad stage and was followed by primexine formation. The small vesicles and vacuoles in close contact with the plasmalemma (Fig. 4) at the tetrad stage indicate that materials deposited at the special sites of the primexine are produced by the microspore. At the free microspore stage, the exine was
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well defined, with the formation of the ektexine (tectum, supratectal elements, columellae and foot layer) and endexine. The tectum was observed dissected with a few microchannels (Fig. 6a). In wheat, the tectum is observed as discontinuous and interrupted with radially oriented microchannels (ElGhazaly and Jensen 1986). However, microchannels were not identified in the foot layer of B. distachyon in the present study. The presence of more microchannels in the tectum than in the foot layer is a general observation for members of Poaceae (Skvarla and Larson 1966; Christensen et al. 1972; Heslop-Harrison 1979; Marquez et al. 1997b). In rice and maize, the presence of numerous microchannels, in both the tectum and the foot layer, has been reported; however, in the same study, the pennisetum pollen wall showed the absence of microchannels in the tectum and the foot layer (Fu et al. 2001). Many authors (Rowley 1973; El-Ghazaly and Jensen 1985) have considered these tectal microchannels to be adaptive mechanisms that help in the interchanging of materials from the inside to the outside of the pollen grain. However, Lin et al. (1979) believed that microchannels are related to the release of proteins and pollen grain recognition by the stigmas. The formation of the tectum begins during the tetrad stage of pollen development in Triticum (El-Ghazaly and Jensen 1986), Lilium (Heslop-Harrison 1971) and Zea (Skvarla and Larson 1966). In Sorghum (Christensen et al. 1972), however, the tectum appears to develop only after the microspores are released from the callose wall. A similar ontogenetic timing for tectum development was observed in B. distachyon during the free microspore stage of development. The endexine develops as a rudimentary, thin layer on the proximal surface of the foot layer during the free microspore stage of development. In B. distachyon, the intine is the last layer to be laid down. In the next stage, during vacuolation, the endexine is pressed against the proximal surface of the foot layer after the formation of the Z-layer, making it difficult to observe. The peripheral cytoplasm at the vacuolate stage of development is dense with Golgi vesicles and dictyosomes, and the vesicles fuse with the plasmalemma, possibly contributing to the formation of the intine (Fernandez and Rodriguez-Garcia 1989). As observed in earlier studies, the distal face of the intine is formed by material transported in Golgi vesicles (Echlin and Godwin 1969; Roland 1971). Z-layer formation is the first contribution to the formation of the intine, though the well developed intine wall is observed after mitosis, during the bicellular pollen stage. A similar timing of intine development has been reported for Triticum (El-Ghazaly and Jensen 1986), Zea (Skvarla and Larson 1966) and Sorghum (Christensen et al. 1972). However, in Lilium, Silene (Heslop-Harrison 1963, 1968a) and Arabidopsis (Owen and Makaroff 1995), the intine develops before mitosis during the free microspore stage of development. Another characteristic feature in the development of the intine is the number of strata it contains. In Poaceae
(Marquez et al. 1997a, b) and Euphorbiaceae (Suarez-Cervera et al. 2001), three layers of intine have been reported. However, in some species, such as Nelumbo lutea (Kreunen and Osborn 1999), Hypecoum imberbe (Romero et al. 2003) and Tarenna gracilipes (Vinckier and Smets 2005), two strata have been reported. Furthermore, a single homogenous stratum has been described in Olea europaea (Fernandez and Rodriguez-Garcia 1988) and Lycopersicon esculentum (Fernandez et al. 1992). In the present study, TEM observations of the mature pollen wall of B. distachyon revealed the presence of a three-layered intine wall. In the current study, we have described the pollen ontogenic sequence in B. distachyon, including the ultrastructure of the mature pollen wall. In particular, the pattern of microsporogenesis, exine formation and aperture development were evaluated for the first time in this model plant. Developmental timing of exine components and tectal microchannels supports the hypothesis that these elements of the exine function in pollenkitt storage. Moreover, absence of microchannels in the foot layer of B. distachyon also illuminates ontogenic differences between B. distachyon and its close member wheat, which reflect reproductive adaptations with evolution. Acknowledgments We thank Dr. Simon Crawford for technical guidance with electron microscopy and access to Advanced Microscopy Facility, The University of Melbourne. AS also thanks Dr. Martin O’Brien for helping in identifying the stages of anther and pollen development and Dr. Lim Chee Liew for suggesting improvements in the manuscript and helping in organizing the figures during manuscript preparation. Financial support from the Australian Research Council (ARC DPO988972) is also gratefully acknowledged. Conflict of interest The authors declare that they have no conflict of interest.
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