Annals of Botany 119 (6): 989-1000 (2017)
1 Original article
2 Title:
3 POLLEN TUBE ACCESS TO THE OVULE IS MEDIATED BY GLYCOPROTEIN
4 SECRETION ON THE OBTURATOR OF APPLE (MALUS X DOMESTICA, BORKH)
5 Authors:
6 Juan M. Losada1,2,3,4*
7 María Herrero4
8 1 Arnold Arboretum of Harvard University. 1300 Centre St. Boston, MA 02131.
9 2 Department of Organismic and Evolutionary Biology, Harvard University. 16 Divinity Ave,
10 Cambridge, MA 02138.
11 3 Department of Ecology and Evolutionary Biology, Brown University. 80 Waterman Street
12 Providence, RI 02912.
13 4Pomology Department, Aula Dei Experimental Station-CSIC. Avda Montañana 1005. 50059.
14 Zaragoza, Spain.
15 Running title: Secretions of the apple obturator
16 *E-mail address: [email protected]
17
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Annals of Botany 119 (6): 989-1000 (2017)
1 ABSTRACT
2 Background and Aims Within the ovary, the obturator bridges the pathway of the pollen tube
3 from the style to the ovule. Despite its widespread presence among flowering plants, its function
4 has been only studied in a handful of species and the molecules involved in pollen tube-obturator
5 crosstalk have not been explored hitherto. This work evaluates the involvement of glucans and
6 glycoproteins on pollen tube growth in the obturator of apple flowers (Malus x domestica).
7 Methods Pollen tube kinetics was sequentially examined in the pistil and related to changes
8 occurring on the obturator using histochemistry and inmunocytochemistry. To discriminate
9 between changes in the obturator induced by pollen tubes from those developmentally regulated,
10 both pollinated and unpollinated pistils were examined.
11 Key Results Pollen tube growth rates were slow in the stigma, faster in the style and slow again
12 in the ovary. The arrival of pollen tubes to the obturator was concomitant with the secretion of
13 proteins, saccharides and glycoprotein epitopes belonging to extensins and arabinogalactan
14 proteins (AGPs). While some of these secretions - extensins and AGPs labeled by JIM13 - were
15 developmentally regulated, others - AGPs labeled by JIM8 - were elicited by the presence of
16 pollen tubes. Following pollen tube passage all these glycoproteins were depleted.
17 Conclusions Results show a timely secretion of glycoproteins on the obturator surface
18 concomitant with pollen tube arrival to this structure. The fact that secretion depletes following
19 pollen tube passage strongly suggests their role in regulating pollen tube access to the ovule.
20 Remarkably, both the regulation of the different glycoproteins secretion, as well as their
21 association with the performance of pollen tubes exhibit similarities with those observed in the
22 stigma, in line with their common developmental origin.
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1 Key words: AGPs, arabinogalactan proteins, apple, extensins, glycoproteins, Malus x
2 domestica, obturator, ovary, pistil, pollen, syphonogamy, transmitting tract.
3
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1 INTRODUCTION
2 From pollination to fertilization, regardless of the angiosperm taxon, pollen tubes travel
3 along three territories: the stigma, the style and the ovary (Heslop-Harrison, 1987, 2000;
4 Dresselhaus and Franklin-Tong, 2013). This apparent complicated scenario offers ample
5 opportunities for pistil support, but also control, of pollen tubes through regulated pistil
6 secretions along the pollen tube pathway (Herrero and Hormaza, 1996; Herrero, 2003).
7 Secretions at the stigma offer adequate support for pollen germination (Herrero and Dickinson,
8 1979; 1981; Hiscock and Allen, 2008, Losada and Herrero, 2012), but this support is not
9 indiscriminate (Hedhly et al, 2005). Along the style, pollen tube elongation relies on provisions
10 stored in the style (Herrero and Dickinson, 1979; Stephenson et al., 2003; Losada and Herrero,
11 2014). But this nutritional dependence also exerts a selective pressure on pollen tubes (Mulcahy,
12 1979; Hormaza and Herrero, 1992, 1994), with clear ecological and evolutionary implications
13 (Lankinen and Green, 2015; Hersh et al., 2015; Harder et al., 2016). When pollen tubes arrive in
14 the ovary they face a territory less well studied. While the molecular control of pollen tubes in the
15 ovary has been extensively studied at the ovules (Higashiyama and Takeuchi, 2015), the ovarian
16 pollen tube pathway between the stylar transmitting tissue and the ovule has remained neglected,
17 except in a handful of species (Arbeloa and Herrero, 1987; Herrero and Arbeloa, 1989;
18 Sornsathapornkul and Owens, 1999; Herrero, 2001, 2003; Nores et al., 2015). Interestingly
19 enough, the few available reports emphasize the obturator as a critical player in pollen tube
20 performance prior to syphonogamy.
21 Aside from the early description of the obturator by Dalmer in 1880, it has been almost
22 entirely overlooked in angiosperms. This is quite surprising, since the obturator has a consistent
23 presence across members of the major clades of flowering plants, including the early diverging
24 genus Kadsura (Schisandraceae; Lyew et al., 2007), mesangiosperm genera such as Persea
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1 (Lauraceae; Sedgley and Anells, 1981), Artabotrys, Magnolia, Liriodendron (Magnoliaceae;
2 Matsui et al., 1993; Igersheim and Endress, 1997), Zygogynum (Winteraceae; Igersheim and
3 Endress, 1997), as well as at least seventy families of monocots and dicots (Stevens, 2001 and
4 refs.; Endress and Mathews, 2006; Nores et al., 2015). Unfortunately, the functionality of the
5 obturator is difficult to evaluate, since it is buried in the ovary and requires a sequential
6 examination of a developmental process. But as a matter of fact, the obturator is a critical
7 territory for pollen tubes as it bridges the stylar transmitting tissue with the ovule entrance (Juel,
8 1919; Tilton and Horner, 1980; Tilton et al., 1984; Endress, 1994). This relationship is
9 straightforward in species with orthotropous ovules in which the ovule entrance faces towards the
10 style (Arbeloa and Herrero, 1987; Herrero and Arbeloa, 1989). In contrast, a sharper switch in
11 directionality occurs in species with anatropous ovules, in which the ovule entrance is opposite
12 that of the style, and pollen tubes have to turn 90º to 180º towards the micropyle (Endress, 2015;
13 Nores et al., 2015). While this directional switch occurs on the obturator surface, its role on
14 pollen tube directionality towards the ovule has scarcely been studied. In peach flowers, pollen
15 tube growth is arrested upon arrival at the obturator until this structure enters a secretory phase,
16 then the pollen tube further resumes growth (Arbeloa and Herrero, 1987). In other species, the
17 obturator enters the secretory phase before pollen tube arrival (González et al. 1996). Still, in all
18 instances examined the provision of a secretion in the obturator appears to be a prerequisite for
19 pollen tube passage, but the timing and nature of this secretion remains enigmatic for most
20 species.
21 Glycoproteins are a major player in the communication between developing pollen tubes
22 and pistil tissues in angiosperms, especially as a form of pollen tube recognition within the style
23 (McClure et al., 2011; Pereira et al., 2015). In Nicotiana, pollen tube nourishment has been
24 further demonstrated by a deglycosylation gradient of glycoproteins along the transmitting tract
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1 as pollen tubes elongate through, consistent with the idea of a basipetal maturation of the pistil
2 (Wu et al., 1995; 2000). Among glycoproteins, arabinogalactan proteins (AGPs) ubiquitously
3 localize in the pollen tube pathway of angiosperms (Cheung et al., 1995; Sánchez et al, 2004;
4 Sage et al., 2009; Pereira et al., 2014). Indeed, they play key roles during the initial stages of the
5 progamic phase (Losada and Herrero, 2012; Suárez et al., 2013; Losada et al., 2014). Curiously,
6 species with large styles such as apple exhibit a variable glycoprotein topology along the pistil. In
7 the stigma of the apple flower, AGPs set the time for pollen receptivity (Losada and Herrero,
8 2012). Conversely, in the contiguous transmitting tract of the style, glycoproteins extensins and
9 preformed beta glucans preclude a rapid pollen tube elongation (Losada and Herrero, 2014). This
10 is where glycoprotein topology analysis stops, leaving the adjacent tissues of the ovary largely
11 unexplored. The concurrent participation of AGPs along other stretches of the pollen tube
12 pathway suggests that they could well be a component of the obturator secretion. Therefore, this
13 work evaluates pollen tube growth in relation to changes in the obturator of the apple (Malus x
14 domestica) flower, with emphasis on the immunolocalization of glycoproteins.
15
16 MATERIALS AND METHODS
17 Plant material
18 Flowers from apple trees cv Golden Delicious Spur grown in the province of Huesca
19 (Spain), 461m above sea level were collected over two years. One day before anthesis (flower
20 opening), flowers were emasculated and left for 24h until hand pollination. Since apples are self-
21 incompatible, anthers from the compatible cv Royal Gala were collected from flowers at the same
22 developmental stage and left to dry on paper at room temperature (20-25ºC) for 24-48h until
23 dehiscence. Pollen was sieved through a metallic mesh with a diameter pore of 0.26 mm, and
24 used to pollinate emasculated flowers, whereas leaving a second batch of flowers unpollinated.
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1
2 Observation of pollen tube growth
3 In order to monitor the arrival of pollen tubes to the different tissues along the pistil,
4 gynoecia were fixed daily from anthesis to seven days after pollination (DAP). Five gynoecia
5 from each batch were fixed in FAA (formalin: acetic acid: 70% ethanol) (1:1:18) (Johansen,
6 1940) for at least five hours. The pistils were then washed in distilled water thrice 1h each, placed
7 in 5% sodium sulphite for 24h, and finally autoclaved for 10min at 1kg cm−2 (Jefferies and
8 Belcher, 1974). Afterwards, the individual styles were dissected and squashed onto glass slides
9 with 0.1% aniline blue in 0.1 NK3PO4 to visualize callose (Currier, 1957) and pollen tubes
10 (Linskens and Esser, 1957). Observations were done with a LEICA DM2500 fluorescence
11 microscope with a 340/400 nm filter, and a CANON Power Shot S50 camera with a CANON
12 Remote Capture software.
13
14 Histochemistry
15 Three pollinated and unpollinated receptacles (30 ovaries) were selected for histochemical
16 preparation at the developmental stages of anthesis, three, six, seven, and ten days later.
17 Receptacles were fixed overnight in 4% paraformaldehyde (w/v) made from fresh formaldehyde,
18 washed in 1X phosphate saline buffer (PBS) three times 15min each, and then kept in 0.1%
19 paraformaldehyde at 4C until use (Solís et al., 2008). Receptacles were then dehydrated through a
20 series of increasing ethanol concentrations (30%, 50%, 70%, 85%, 95%, 100%) for one hour in
21 each, and then embedded in Technovit8100 glycol methacrylate resin (Kulzer and Co, Germany).
22 They were sectioned at 2μm with a LEICA EM UC6 ultramicrotome and placed onto slides
23 previously coated with 2% (3-Aminopropyl)-triethoxysilane (Sigma-Aldrich).
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1 To identify insoluble carbohydrates, sections were stained with Periodic Acid Shiffs (PAS)
2 (Feder and O’Brien, 1968). In order to stain for proteins, a solution of 0.25% aniline blue black in
3 1% acetic acid (v/v) (Fisher, 1968) was used.
4
5 Inmunocytochemistry
6 Gynoecia, fixed as above in 4% paraformaldehyde and similarly sectioned, were used for
7 inmunocytochemistry. Immunolocalization of callose on the obturator was carried out with the
8 use of the anti-β-(1,3)-glucan antibody (Meikle et al., 1991). To investigate glycoprotein content,
9 three monoclonal antibodies were further used to detect each specific epitope: JIM11 mAb for
10 extensins (Smallwood et al., 1994; Yates et al., 1994), JIM13 and JIM8 for arabinogalactan
11 protein glycosyl epitopes (Knox et al., 1991; Pennell et al., 1991), using a procedure previously
12 described for apple pistil tissues (Losada and Herrero, 2012; 2014). Following
13 inmunolocalizations, sections were counterstained for cellulose and some other pectic compounds
14 with 0.07% calcofluor white (v/v) (Hughes and McCully, 1975; Coimbra et al., 2007), mounted
15 in PBS or Mowiol, and examined with a Leica DM2500 microscope with epifluorescence,
16 355/455 nm filter was used for calcofluor white, and a 470/525 nm one for the Alexa 488
17 fluorescein label of the secondary antibodies.
18
19 RESULTS
20 Pollen tube growth
21 Pollen grains germinated at the stigma and traversed 0.75mm along two days before
22 entering the style (Fig. 1A). Once in the stylar transmitting tissue, pollen tube growth rate
23 increased, and pollen tubes covered a tenfold distance of 10mm over two days. The elongation
24 rate of pollen tubes slowed down at the ovary, where a much shorter distance was covered in two
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1 more days, prior to fertilization which happened the seventh day after pollination. Upon closer
2 observations of the pollen tubes, various shapes were noted on their walls at the three areas of the
3 pistil traversed: first, a wandering pattern on the stigma surface (Fig. 1B); second, a more
4 directional pollen tube wall formation and elongation within the style (Fig. 1C); finally, a jerky
5 pollen tube wall shape on the obturator surface before ovule penetration (Fig. 1D).
6
7 Developmental changes in the obturator before and after pollen tube arrival
8 The pentacarpelar apple gynoecium contains five ovary locules, each one accommodates
9 two anatropous ovules which face two corresponding obturators (Fig. 2A). In longitudinal
10 section, the obturator appeared as a funicular protuberance facing the ovule exostome (Fig. 2B).
11 While pollen tubes elongate downwards between the funiculus and the placental tissue, they
12 sharply turn 180º on the obturator surface before entering the micropyle (Fig. 2C).
13 At anthesis (ANTH), the papillated cells of the obturator surface were highly vacuolated
14 and their cytoplasms reacted for proteins (Fig. 3A), and stored polysaccharides (Fig. 3B). In
15 unpollinated pistils, seven days after anthesis (7DAA), proteins continued to be present within
16 the obturator cells (Fig. 3C), but the papillar cell walls enlarged as insoluble polysaccharides
17 were conspicuously present in the cells of the obturator surface (Fig. 3D). In contrast, a secretion
18 that stained positive for proteins appeared on the obturator surface concomitant with pollen tube
19 passage, in pollinated flowers seven days after pollination (7DAP) (Fig. 3E). Additionally,
20 polysaccharides vanished from cells on the obturator, while starch was clearly present in the
21 growing pollen tubes (Fig. 3F). Since proteins and carbohydrates were detected in these
22 secretions, the possible involvement of glycoproteins was further explored.
23
24 Glycoproteins and callose in the obturator
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Annals of Botany 119 (6): 989-1000 (2017)
1 At anthesis, epitopes labelled by JIM13 mAb had a discrete presence inside the obturator cells
2 (Fig. 4A). Three days later, they appeared on the obturator surface in both unpollinated and
3 pollinated pistils before pollen tube arrival (Fig. 4B). In unpollinated flowers this secretion
4 remained six days after anthesis (Fig. 4C). In pollinated flowers, upon pollen tube arrival at the
5 obturator, secretions labeled with JIM13 were surrounding the pollen tubes and further mapped
6 onto areas previously identified as starch grains (Fig. 3D, 4D). While unpollinated flowers
7 continued to show JIM13 epitopes on the obturator surface ten days after anthesis (10DAA), (Fig.
8 4E), in pollinated flowers they entirely vanished from the obturator surface, following pollen tube
9 passage (Fig. 4F).
10 In contrast, secretion of AGPs recognized by JIM8 mAb showed a different secretory
11 pattern. JIM8 epitopes were barely present within cells of the obturator at anthesis (Fig. 5A), but
12 their detection increased three days later (Fig. 5B). Six days after anthesis, JIM8 epitopes were
13 not observed on the obturator surface in unpollinated pistils (Fig. 5C). Whereas in pollinated
14 flowers, pollen tube arrival triggered the secretion of JIM8 epitopes to the obturator surface, (Fig.
15 5D). Ten days after anthesis, JIM8 epitopes were confined within the cells of the obturator in
16 unpollinated pistils associated to starch grains (Fig. 5D, 5E), while these epitopes were strikingly
17 depleted from the obturator following pollen tube passage in pollinated pistils, following a
18 similar pattern of that observed for polysaccharides (Fig. 5F).
19 Immunolocalization of callose epitopes exhibited a weak localization in the cytoplasm of
20 the obturator cells at anthesis (Fig. 6A). Similarly, extensin epitopes were almost undetectable in
21 the obturator at flower opening (Fig. 6B). The arrival of pollen tubes to the obturator surface was
22 clear by the presence of callose in the pollen tube wall but not in the obturator (Fig. 6C). In turn,
23 extensins were detected close to the pollen tube walls on the obturator surface of pollinated pistils
24 (Fig. 6D), but also their immuno detection on the obturator surface of unpollinated pistils
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1 suggested a developmental secretion (Fig. 6D, inset). While callose was still profusely present in
2 the walls of pollen tubes, ten days after pollination (10DAP) (Fig. 6E), it was not observed in the
3 obturator cells of pollinated nor unpollinated pistils (Fig. 6E, inset). Simultaneously, more pollen
4 tubes arrived at the obturator surface and showed contact with extensin epitopes of the obturator
5 surface (Fig. 6F). Oddly enough, these epitopes were weakly detected in unpollinated obturators
6 (Fig. 6F, inset).
7
8 DISCUSSION
9 This work evidences the timely secretion of glycoproteins towards the surface of the
10 obturator concomitantly with pollen tube arrival, suggesting a pivotal role of the obturator prior
11 to syphonogamy.
12
13 A timely secretion in the obturator
14 Results herein show that secretions of the obturator occur at a particular time in
15 development, which precludes pollen tube arrival at that structure. While much attention has been
16 given to the gametophytic control of pollen tube attraction in model species (Higashiyama and
17 Hamamura, 2008; Márton and Dresselhauss, 2010; Mizukami et al., 2016), the sporophytic
18 pathway within the ovary has received much less attention. Yet, in non-model species, works
19 have highlighted that syphonogamy is precluded by several checkpoints within the ovarian
20 chamber in both dicots (Herrero, 2000, 2001, 2003; Sogo and Tobe, 2005, 2006; Distefano et al.,
21 2011), and monocots (Márton et al., 2005; Lausser et al, 2010). The obturator is the sporophytic
22 structure pollen tubes traverse prior to ovule penetration, but it has received little attention
23 (Arbeloa and Herrero, 1987; Herrero and Arbeloa, 1989; Sornsathapornkul and Owens, 1999;
24 Herrero, 2001, 2003; Nores et al., 2015). Our observations show that polysaccharides are
11
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1 secreted towards the surface of the obturator prior to pollen tube arrival, which are further
2 depleted following the passage of pollen tubes. Secretions from the obturator have been directly
3 related to pollen tube growth towards the ovule (Arbeloa and Herrero, 1987; Herrero, 2000,
4 2003). But while the secretory nature of the obturator has been reported in a few plant taxa, it is
5 noteworthy that the timing of this secretion varies across species. For example, in kiwifruit
6 flowers, copious exudates were present on the obturator right from flower opening (González et
7 al., 1996). On the contrary, secretions towards the obturator surface of Prunus persica took place
8 12 days after anthesis, long after pollen tubes have arrived at this structure that remained waiting
9 for this secretion to continue their way towards the ovule (Arbeloa and Herrero, 1987),
10 suggesting a key control of these secretions over pollen tube growth.
11 The concomitance of secretions from the obturator and pollen tube arrival has also been
12 reported in a few angiosperm genera, such as Rhododendron (Palser et al., 1989), Ornithogalum
13 (Tilton and Horner, 1980), and Phellodendron (Zhou et al., 2004). In the absence of an obturator,
14 similar structures within the ovary chamber, such as the hair-like cells in the placenta of Zea
15 mays (Heslop-Harrison et al., 1985), the papillae in Citrus reticulata (Distefano, et al., 2011) or
16 the glandular hairs of Annona cherimola (Lora et al., 2010), exhibit a similar secretory pattern,
17 pointing to the secretions of the ovarian sporophytic tissues as major players in pollen tube
18 performance, before ovule entrance, in most flowering plants. While the secretory function of the
19 obturator appears to be constant in whatever species it has been investigated, the molecular nature
20 of these secretions remains elusive.
21
22 Secretion of glycoproteins mediate obturator receptivity
23 Our results in apple flowers reveal that the secretion of glycoproteins mediate obturator
24 receptivity, a concept analogous to stigma receptivity raised from the current results. While
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1 secretions composed of saccharides have been reported in the obturator of a number of species
2 (Tilton and Horner, 1980; Arbeloa and Herrero, 1987), the molecular composition of this
3 secretion has not been examined in any angiosperm thus far.
4 Two arabinogalactan proteins with different glycosyl epitopes are secreted to the apple
5 obturator surface in different patterns: AGPs with epitopes recognized by JIM13 mAb are
6 developmentally secreted, whereas AGPs detected by JIM8 mAb are elicited by the pollen tubes.
7 JIM13 epitopes could not be detected at anthesis, but secretion starts three days after anthesis
8 with a massive presence on the obturator surface concomitantly with pollen tube arrival at the
9 obturator. In unpollinated pistils, this secretion was also produced at the same time, suggesting a
10 developmental control of this secretion. A similar situation for these epitopes has been reported
11 in the apple stigma, where the developmental secretion of JIM13 epitopes marks the acquisition
12 of stigmatic receptivity (Losada and Herrero, 2012). The presence of JIM13 epitopes has also
13 been reported in the stigma of olive (Suárez et al., 2013), and banana (Sharma and Bhatla, 2013)
14 flowers at anthesis. Additionally, work in the protogynous flowers of the relatively early
15 diverging genus Magnolia (Losada et al., 2014) highlighted the association of JIM13 epitopes
16 with its short stigmatic receptivity, suggesting the conservation of this epitope in the receptive
17 stigma of a wide range of angiosperms. Interestingly, our results point to a subsequent depletion
18 of JIM13 epitopes from the obturator surface, suggesting that growing pollen tubes utilize these
19 epitopes, a relationship that is quite similar to that reported for the stigma (Losada and Herrero,
20 2012, Losada et al. 2014).
21 Yet, another set of AGPs is detected with JIM8 mAb in the obturator of apple. Secretion
22 of these arabinogalactans are elicited towards the obturator surface by the arrival of the pollen
23 tubes, given that they are not secreted in unpollinated flowers. Strikingly, the same pattern was
24 reported to occur in the stigma of apple flowers (Losada and Herrero, 2012). In both the obturator
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1 and the stigma of apple, secretion of JIM8 epitopes to the surface of the papillated cells are
2 induced by pollen tube arrival, and then deplete from these tissues as pollen tubes pass by. The
3 fact that JIM8 epitopes accumulate within cells of the obturator in unpollinated pistils strongly
4 suggests that their secretion is elicited by the presence of pollen tubes.
5 Although the lack of immunolocalization studies in the obturator of other angiosperm
6 species hampers a comparative framework to be completed, our results suggest developmental
7 similarities between the stigma and the obturator.
8
9 A shared secretory pattern between the obturator and the stigma
10 The secretory function of the apple pistil correlates well with pollen tube growth rates
11 along the three different territories traversed: slow in the stigma, fast in the style and slow again
12 within the ovary. Secretions of the stigma mainly belong to two AGPs epitopes, JIM8 and JIM13
13 (Losada and Herrero, 2012). In turn, AGPs have not been detected in the contiguous stylar
14 transmitting tissue, but profuse extensins and callose epitopes associate with an acceleration of
15 pollen tube growth rate (Rubinstein et al., 1995; Losada and Herrero, 2014). Interestingly, while
16 the obturator further secretes extensins in a developmental fashion, AGPs appear to only account
17 for pollen tube support in this tissue.
18 The secretory pattern of AGPs from the obturator resembles that of the stigma, and may
19 contributes to the picture building with the existing molecular works on the secretome of the
20 stigma (Rejon et al., 2013, 2014), and the ovary (Liu et al., 2015). Indeed, a deceleration of the
21 pollen tube growth rate occurs in the ovary, together with a jerky construction of the pollen tube
22 walls on the obturator surface, prior to ovule penetration. This uneven appearance of pollen tubes
23 prior to ovule penetration has also been reported in other species such as Prunus (Herrero, 2000,
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1 2001) or Fagus (Sogo and Tobe, 2006), and slowdowns of pollen tube growth appear to be
2 common (Herrero and Arbeloa, 1989, Herrero, 2003). This effect could be enhanced by the role
3 of endogenous AGPs produced by the pollen tube walls (Tan et al., 2010; Costa et al., 2015;
4 Lopes et al., 2016), and point to a certain degree of pollen tube wall plasticity depending on the
5 medium in which it elongates (Castro et al., 2013; Losada and Herrero, 2014).
6 Certainly, both stigma and obturator appear to be spatially separated secretory tissues, and
7 the different timing taken to enter a secretory phase likely responds to the basipetal maturation of
8 the pistil (Herrero and Arbeloa, 1989; Herrero, 1992; Losada and Herrero, 2014). A basipetal
9 maturation synchronizes with the development of the male gametophyte along the pistil of the
10 apple flower, as it occurs in other species (Herrero, 2003). As a result, the switch from the stylar
11 transmitting tract to the obturator displays variation in the strategic provisioning of pollen tubes.
12 This is in line with the idea that evolving the style prolonged pollen pistil interactions in
13 angiosperms (Lora et al, 2016), favouring gametophyte competition and selection (Mulcahy,
14 1979; Hormaza and Herrero, 1992, 1996). All together, these data suggest that the secretory
15 program is tightly regulated in the apple pistil, but also highlights functional similarities between
16 the stigma and the obturator.
17 The similarities of glycoprotein secretion in both the obturator and stigma might relate to
18 the common ontogenic origin of both structures. As with the stigma, the obturator derives from
19 the inner epidermis, which forms the suture line during carpel closure. After development of the
20 carpel, the stigma faces outwards, whereas the obturator is buried in the ovary. Interestingly, the
21 distance between the stigma and the obturator increases from early divergent angiosperms to
22 more recently derived clades (Endress, 2000; Lora et al., 2010; 2016). Although three different
23 developmental origins have been reported for the obturator within the ovary (Endress 2015), it
15
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1 clearly evolved several times independently among angiosperms (Endress and Matthews, 2006).
2 Indeed, it has been described so far in six orders of monocots and seventeen orders of eudicots
3 (Stevins, 2001 and refs.), therefore suggesting that the evolutionary origins of the obturator
4 associate with angiospermy. This is further supported by the fact that the only gymnosperm
5 described as having an obturator is Gnetum (Strasburger, 1872; Berridge, 1911), but its location
6 as an outgrowth of the integuments sealing the micropyle at the pollen tube pathway (Waterkeyn,
7 1960) calls into question its possible homoplasy with the angiosperm obturator.
8 Nevertheless, the obturator has been traditionally seen as a compensatory tissue without
9 specific functionality. Results in apple flowers show that the obturator does play a clear part
10 regulating pollen tube passage and access to the ovules, through the regulated secretion of AGPs.
11 It would be interesting to evaluate how conserved the secretion of glycoproteins on the obturator
12 appears to be among angiosperms prior to fertilization.
13
14 ACKNOWLEDGEMENTS
15 Authors are very grateful to Reyes Lopez for technical assistance and Grace Yu for her help with
16 English edits. This work was supported by Ministerio de Ciencia e Innovación (MICINN)-
17 FEDER [AGL2006-13529-C02-01, AGL 12621-C02-01, AGL 2012–40239], and Gobierno de
18 Aragón [group A43]. JIM antibodies distribution was partly supported by NSF grants [DBI-
19 0421683, RCN 009281]. J.M.L. was supported by a FPI fellowship [BES-2007-16059] from
20 MICINN.
21
22 LITERATURE CITED
23 Arbeloa A, Herrero M. 1987. The significance of the obturator in the control of pollen tube
24 entry into the ovary in peach (Prunus persica). Annals of Botany 60: 681–685.
16
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1 Berridge EM. 1911. On some points of resemblance between Gnetalean and Bennettitean seeds.
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3 Castro, A.J., Suárez, C., Zienkiewicz, K., de Dios Alché, J., Zienkiewicz, A. and Rodríguez-
4 García, M.I. 2013. Electrophoretic profiling and immunocytochemical detection of pectins and
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20
21 FIGURE LEGENDS
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1 Figure 1. Pollen tube growth rate along the apple pistil. A. Pollen tube kinetics showing three
2 different pollen tube growth rates depending on the tissue of the pistil traversed: slow in the
3 stigma (blue), fast in the style (yellow), and slow again in the ovary (blue). B. Detail of the pollen
4 tube wall construction in the stigma showing a wandering pattern. C. Straight pollen tube wall
5 formation within the style. D. Jerky pollen tube wall (arrows) on the obturator surface within the
6 ovary. B, C, D: whole mounts of hand pollinated apple pistils stained with aniline blue. Scale
7 bars: 10µm.
8
9 Figure 2. Anatomy of the obturator in the apple ovary. A. Two anatropous ovules of apple with
10 each exostome facing an obturator. B. Median longitudinal section of an ovule stained for cell
11 walls showing the pollen tube pathway before fertilization. C. Outline of the median section of an
12 apple ovary showing the pollen tube pathway (blue), which turns 180 degrees on the obturator
13 surface towards the ovule. Abbreviations: fg: female gametophyte; funic: funiculus; ii: inner
14 integument; nuc: nucellus; obt: obturator; oi: outer integument; plac: placenta. Scale bars:
15 100µm.
16
17 Figure 3. Developmental changes in the obturator of apple. A. At anthesis (ANTH), protein
18 contents (stained in blue) of the obturator cells were mainly localized in the cytoplasm. B. Cells
19 of the obturator surface show starch (red arrows) at anthesis. C. In unpollinated pistils, seven
20 days after anthesis (7DAA), cells of the obturator surface are vacuolated and cytoplasms
21 profusely stain for proteins. D. Insoluble polysaccharides within cells of the obturator surface
22 (red arrows) of unpollinated pistils, but wider cell walls on the surface. E. In pollinated pistils, the
23 arrival of pollen tubes elicited secretion of proteins towards the obturator surface (black arrows).
24 F. Polysaccharides vanished from the obturator cells, as they were present in pollen tubes (red
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1 arrows). 4µm sections of Technovit8100 embedded apple ovaries stained with aniline blue black
2 for proteins (A, C, E), and PAS-Shiffs for polysaccharides (B, D, F). Abbreviations: pt: pollen
3 tube; ANTH: anthesis; DAA: days after anthesis; DAP: days after pollination. Scale bars: 20µm.
4
5 Figure 4. Changes of AGPs recognized by the JIM13 monoclonal antibody (mAb) in the apple
6 obturator. A. JIM13 epitopes (labelled with fluorescent green) within cells of the obturator at
7 anthesis (ANTH). B. JIM13 epitopes secreted to the obturator surface (arrows) before pollen tube
8 arrival three days after pollination (3DAP). C. JIM13 labelled arabinogalactans on the obturator
9 surface in unpollinated pistils (arrows) six days after anthesis (6DAA). D. JIM13 epitopes
10 associated to the pollen tube wall (asterisks) on the obturator surface. E. JIM13 epitopes
11 profusely present on the obturator surface of unpollinated pistils (arrows) ten days after anthesis
12 (10DAA). F. Ten days after pollination (10DAP) JIM13 epitopes completely vanished from the
13 obturator surface. 4µm sections of Technovit8100 embedded apple ovaries immunolocalized with
14 JIM13mAb, and recognized by a secondary antibody conjugated with a FITC molecule, which
15 emits green fluorescence. Calcofluor white (blue colour) is used to counterstain cellulose and
16 pectins of the cell walls. Abbreviations: pt: pollen tube; ANTH: anthesis; DAA: days after
17 anthesis; DAP: days after pollination. Scale bars: 20µm.
18
19 Figure 5. Changes of AGPs recognized by the JIM8 monoclonal antibody (mAb) in the apple
20 obturator. A. Weak detection of JIM8 epitopes (fluorescent green) in the obturator at anthesis
21 (ANTH). B. JIM8 epitopes labelled within cells of the obturator three days after pollination
22 (3DAP). C. JIM8 epitopes were not secreted to the obturator surface of unpollinated pistils six
23 days after anthesis (6DAA). D. In contrast, JIM8 epitopes were clearly detected on the cells of
24 the obturator surface (arrows), concomitantly with pollen tube arrival six days after pollination
28
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1 (6DAP). E. JIM8 epitopes within cells of the obturator of unpollinated pistils ten days after
2 anthesis (10DAA). F. In pollinated pistils (10DAP) labelling of epitopes was no longer detected
3 in the obturator cells, as plugs of cellulose accumulated on the obturator surface (asterisk). 4µm
4 sections of Technovit8100 embedded apple ovaries immunolocalized with JIM8mAb, recognized
5 by a secondary antibody conjugated with a FITC molecule, which emits green fluorescence.
6 Calcofluor white (blue colour) is used to counterstain cellulose and pectins of the cell walls.
7 Abbreviations: pt: pollen tube; ANTH: anthesis; DAA: days after anthesis; DAP: days after
8 pollination. Scale bars: 20µm.
9
10 Figure 6. Changes of callose and extensins in the obturator of apple. A. Weak detection of
11 callose epitopes in cells of the obturator cells at anthesis (ANTH). B. Extensin epitopes were not
12 labelled in the obturator at anthesis. C. Pollen tube walls on the obturator surface showed strong
13 labelling of callose, but these beta glucans were not detected in the obturator. D. Detection of
14 extensins at intercellular spaces of the obturator (white arrows) at the time of pollen tube arrival
15 six days after pollination (6DAP), as well as conspicuous presence of extensins on the surface of
16 unpollinated obturators six days after anthesis (6DAA, inset). E. Callose was conspicuously
17 labelling pollen tube walls, but undetected in cells of both pollinated and unpollinated obturators,
18 ten days after anthesis (10DAA, inset). F. In contrast, extensin epitopes were present on the
19 obturator surface (arrows) after pollen tube passage, ten days after pollination (10DAP), whereas
20 undetected in unpollinated pistils ten days after anthesis (10DAA, inset). 4µm sections of
21 Technovit8100 embedded apple ovaries immunolocalized with either an anti-(1-3; 1-4)-beta-
22 glucans (A, C, E) or JIM11mAb (B, D, F) antibodies, recognized by a secondary antibody
23 conjugated with a FITC molecule, which emits green fluorescence. Calcofluor white (blue
29
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1 colour) is used to counterstain cellulose and pectins of the cell walls. Abbreviations: pt: pollen
2 tube; ANTH: anthesis; DAA: days after anthesis; DAP: days after pollination. Scale bars: 20µm.
3
4 Figure 7. Glycoprotein distribution in the apple pistil during the progamic phase. Outlines of a
5 median longitudinal section of an individual apple pistil at different developmental stages from
6 anthesis (ANTH) to the time of fertilization six days after pollination (6DAP). A. JIM13 epitopes
7 (green) are developmentally secreted to the apple stigma before pollination. B. Pollen tube
8 elongation in the stigma lasted for two days, and induced secretion of JIM8 epitopes (green) to
9 the stigmatic surface, while JIM11 epitopes (extensins) were accumulating on the top area of the
10 stylar transmitting tract (red). C. Pollen tubes elongate along the apple pistil for two more days,
11 and both extensin and callose epitopes were accumulated in the transmitting tissue of the style in
12 a basipetal fashion; concomitantly, JIM13 and JIM11 epitopes (green and red) were secreted
13 towards the obturator surface. D. Pollen tube elongation through the obturator triggered secretion
14 of JIM8 epitopes, which vanished along with JIM13 and JIM11 epitopes following pollen tube
15 passage.
30