Losada JM, Herrero M, Hormaza JI and Friedman WE 1
1 Losada et al. Stigma receptivity and AGPs in Magnolia
2
3 ARABINOGALACTAN PROTEINS MARK STIGMA RECEPTIVITY IN THE
4 PROTOGYNOUS FLOWERS OF Magnolia virginiana (MAGNOLIACEAE)1
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6 Juan M. Losada2,3, Maria Herrero4, Jose I. Hormaza5, William E. Friedman2,3
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8 2Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford
9 Street, Cambridge, Massachusetts 02138 USA; 3Arnold Arboretum of Harvard
10 University. 1300 Centre Street, Boston, Massachusetts 02131 USA;
11 4Department of Pomology, Aula Dei Experimental Station-CSIC. Avda. Montañana
12 1005, Zaragoza, Spain 50059; 5Department of Subtropical Fruits, Instituto de Hortofruticultura
13 Subtropical y Mediterránea “la Mayora,” (IHSM la Mayora–CSIC–UMA). Algarrobo–Costa,
14 Málaga, Spain 29750.
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17 Losada JM, Herrero M, Hormaza JI and Friedman WE 2
1 Acknowledgements
2 JML has been supported by a FPI fellowship from the Spanish Ministry of Science and
3 Innovation, a Deland Award for student research (summer 2011) and a Putnam Scholar
4 (summer 2012), both from the Arnold Arboretum of Harvard University. Research
5 supported in part by NSF grants to WEF [DBI-0421683, RCN 009281] and by grants from
6 the Spanish Ministerio de Economía y Competitividad to MH [AGL2012-40239] and JIH
7 [AGL2013-43732-R].
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22 1Manuscript received ______; revision accepted______.
23 3e-mail for correspondence: [email protected] Losada JM, Herrero M, Hormaza JI and Friedman WE 3
1 ABSTRACT
2 Premise of the study: Factors affecting flower receptivity in angiosperms remain opaque,
3 but recent studies suggest that the acquisition of stigma receptivity associated with cell
4 wall related arabinogalactan proteins (AGPs) may be a widespread feature of flowering
5 plants. Here, the time during which a stigma is receptive is evaluated and related to the
6 secretion of AGPs in Magnolia virginiana, a protogynous member of an early divergent
7 angiosperm clade (magnoliids) with a clearly discernible female receptive phase.
8 Methods: Magnolia virginiana flower phenology was documented and histochemical
9 changes in the stigma before and after pollination were examined. Stigma receptivity was
10 evaluated in relation to the secretion of AGPs detected in whole mounts and
11 immunolocalized in sectioned stigmas.
12 Key results: Protogynous Magnolia flowers display a precise window of stigmatic
13 receptivity, which is concomitant with the secretion of two AGPs labeled for different
14 epitopes. Following pollen germination and tube growth these two AGPs could no longer
15 be detected in the stigmas, suggesting that these AGPs interact with the growing male
16 gametophytes and could be markers of stigma receptivity.
17 Conclusions: These results show that the period of stigmatic receptivity is finely
18 coordinated with the secretion of two arabinogalactans on stigmas of flowers of M.
19 virginiana. This first report of AGP presence in stigmatic tissues in a member of the
20 magnoliids, together with recently described similar patterns in eudicots, monocots, and
21 members of early divergent lineages of flowering plants, suggests an ancient and
22 widespread role for AGPs on stigma receptivity in angiosperms. Losada JM, Herrero M, Hormaza JI and Friedman WE 4
1 Keywords: AGPs; arabinogalactan proteins; Magnolia; pollen; protogyny; receptivity;
2 stigma; stigmatic receptivity.
3
4 INTRODUCTION
5 In spite of the tremendous diversity of flowering plants, the reproductive phase in
6 angiosperms is a highly conserved process in which syngamy and the production of the
7 next sporophyte generation result from a tightly coordinated set of developmental
8 interactions between three individuals: the male gametophyte, the female gametophyte,
9 and the sporophytic tissues of the gynoecium. Many of these interactions take place
10 through plant cell walls that precisely regulate intercellular communication (Martinière et
11 al., 2012). While the vast majority of the molecules that mediate this dialog remain
12 cryptic, it is noteworthy that, among the most ubiquitous components of plant cell walls,
13 pectins and glycoproteins are structural compounds that also may serve as key signaling
14 molecules (Wolf et al., 2012).
15 Among glycoproteins, arabinogalactan proteins (AGPs) are complex hydroxyproline-rich
16 proteins heterogeneously glycosylated with crucial functions at the tissue and cellular
17 levels of organization throughout plant development (Seifert and Roberts, 2007; Ellis et
18 al., 2010; Lamport and Varnai, 2013). During the reproductive process, AGPs are known
19 to be expressed in the male gametophyte, where they are involved in pollen tube
20 development and gametogenesis (Coimbra et al., 2009; 2010; Lin et al., 2014), and also
21 in megaspore development (Demesa-Arévalo and Vielle-Calzada, 2013). But AGPs are
22 also present in sporophytic tissues, especially those involved in the interaction with
23 germinating pollen grains and growing pollen tubes. Thus, AGPs were initially identified Losada JM, Herrero M, Hormaza JI and Friedman WE 5
1 biochemically in the stigmatic exudates of Nicotiana and Lilium at flower opening
2 (Clarke et al., 1979; Gell et al., 1986; Bacic et al., 1988) and, more recently, in Petunia
3 (Twomey et al., 2013). In apple flowers (Malus domestica), acquisition of stigmatic
4 receptivity was shown to be mediated through the secretion of AGPs (Losada and
5 Herrero, 2012).
6 Besides their presence in the early stages of pollen-pistil interactions, AGPs are also
7 present further down along the pollen tube pathway in the style. Work with monoclonal
8 antibodies reactive to specific AGP epitopes has revealed their presence in the gynoecial
9 tissues of very different species from monocots such as Lilium (Jauh and Lord, 1996) to
10 core eudicots such as Arabidopsis (Coimbra et al., 2007), Olea (Suárez et al., 2012), and
11 Nicotiana (Cheung et al., 1995; Wu et al., 2000; Hancock et al., 2005). Indeed,
12 construction of the pollen tube wall within the style appears to require the incorporation
13 of AGPs at different locations along the pollen tube wall (Dardelle et al., 2010).
14 The presence of AGPs in the stigmas, styles and transmitting tissues of monocot and
15 eudicot species with closed carpels contrasts with a relative paucity of information on
16 their possible presence in many of the most ancient flowering plant lineages, some of
17 which are characterized by large stigmas directly connecting to transmitting tissues
18 (Carnright, 1960; Endress and Igersheim, 2000; Endress, 2010). AGPs have been
19 detected in the stigmas of some of the most early-divergent angiosperm clades,
20 Nymphaeales (Trithuria; Prychid et al., 2011), Amborellales (Amborella),
21 Austrobaileyales (Trimenia and Kadsura; Lyew et al., 2007; Sage et al., 2009), and
22 Chloranthales (Sarcandra and Chloranthus; Hristova et al., 2005). Oddly enough, there
23 have been no studies of AGPs in flowers of members of the Magnoliales. Losada JM, Herrero M, Hormaza JI and Friedman WE 6
1 For most of the last century, members of the Magnoliales were thought to be among the
2 most ancient lineages of extant flowering plants. This predominant view was overthrown
3 by the discovery (Mathews and Donoghue, 1999; Qiu et al., 1999, 2010; Barkman et al.,
4 2000; Graham and Olmstead, 2000; Soltis et al., 1999, 2000, 2011; Zanis et al., 2002;
5 Gorekymin et al., 2013) that Amborella, Nymphaeales, and Austrobaileyales comprise
6 three of the four most ancient extant lineages of flowering plants (the fourth clade is all
7 other angiosperms). Around the same time, strong evidence emerged that Magnoliales
8 was part of a larger monophyletic group, the magnoliids that also includes Cannellales,
9 Laurales, and Piperales. Although the exact phylogenetic position of magnoliids within
10 angiosperms remains somewhat uncertain, it appears that this clade is relatively ancient
11 and may predate the origin of monocots and eudicots (Doyle and Endress, 2010, 2014).
12 The fossil record for Magnoliaceae, specifically, is known to extend to the latest
13 Albian/earliest Cenomanian (approximately 100 million years ago) (Dilcher and Crane,
14 1984; Friis et al., 2010), more than 30 million years after the documented origin of
15 flowering plants in the fossil record.
16 Magnolia virginiana L. is one of the over 200 extant species of Magnoliaceae (Kim and
17 Suh, 2013), and is native to the eastern United States. Magnolia flowers are bisexual,
18 with an elongated conical axis where stamens and carpels are arranged spirally. These
19 flowers display many reproductive features that are thought to be relatively
20 plesiomorphic among ancient angiosperms, including beetle pollination (Maneval, 1914),
21 a protogynous flowering cycle (Thien, 1974; Bernhardt and Thien, 1987; Bernhardt,
22 2000), and thermogenicity (Dieringer et al., 1999; Thien et al., 2000). Protogynous
23 dichogamy (with each flower opening twice: first functionally as a female and then next Losada JM, Herrero M, Hormaza JI and Friedman WE 7
1 functionally as a male), plays out in M. virginiana over a period of two days and is
2 critical to preventing selfing (McDaniel, 1966; Wang et al., 2010). Previous reports in M.
3 obovata suggest that stigma receptivity is brief (Wang et al., 2010), but this has not been
4 investigated in M. virginiana L. Virtually nothing is known of the role of glycoproteins
5 during pollen reception and pollen tube growth in this or any other species within the
6 Magnoliales, but this species presents an excellent opportunity to evaluate the association
7 of AGPs with stigmatic receptivity, since it shows a precise easily identifiable female and
8 subsequent male phase. This work evaluates the expression of AGPs during stigma
9 development and pollen-stigma interactions in M. virginiana, with the goal of knowing if
10 these molecules are involved in the first steps of male-female dialogue in this moderately
11 ancient lineage of flowering plants.
12 MATERIALS AND METHODS
13 Plant material — Flowers of Magnolia virginiana (accession numbers 762-83B,
14 `Satellite´145-96A, and `Australis´1275-80B) were photographed and collected at
15 different developmental stages (Fig. 1) during July and August 2011 and 2012 at the
16 Arnold Arboretum of Harvard University (Boston, USA).
17 Pollination experiments — To evaluate stigmatic receptivity, unpollinated flowers and
18 flower buds were collected at different developmental stages, brought to the laboratory
19 and kept in water until the stigmas were pollinated with pollen from male stage flowers.
20 Pollen was applied to flowers collected at the following developmental stages: seven days
21 before first flower opening (-7D), one day before first flower opening (-1D), first day of
22 flower opening (open tepals (0D, 19:00h), closed tepals (0D, 22:00h)), and, after flower Losada JM, Herrero M, Hormaza JI and Friedman WE 8
1 reopening, (1D, 18:00h). For flowers pollinated during the first flower opening, the
2 gynoecia were fixed in FAA (formalin: acetic acid: 70% ethanol) (1:1:18) (Johansen,
3 1940) at two, six and twelve hours after pollination. Following four hours of fixation, the
4 stigmas were washed three times in distilled water 15 min each, and left in 5% sodium
5 sulphite for 24 hours (Jefferies and Belcher, 1974). After that, stigmas were squashed and
6 stained with aniline blue in K3PO4 to observe pollen tubes (Linskens and Esser, 1957),
7 and auramine O in 0.05M phosphate buffer, pH 7.8 (Heslop-Harrison and Shivanna,
8 1977) to visualize cutin and insoluble lipids. Z-axis images were obtained with a Zeiss
9 Confocal Laser Scanning Microscope 700, and 3-D image compositions with the Zeiss
10 LSM software.
11 Histochemical procedures — Both pollinated and unpollinated stigmas were fixed in 4%
12 formaldehyde made from paraformaldehyde in 1X phosphate buffer (PBS) (137mM
13 NaCl, 2.7mM KCl, 10mM Na2HPO4 • 2 H2O, 2.0mM KH2PO4), pH 7.3, then left
14 overnight at 4ºC, and kept in cold at 0.1% formaldehyde in 1X PBS (González-Melendi
15 et al., 2005). Following three washes in 1X PBS, samples were dehydrated in an acetone
16 series (30%, 50%, 70%, 90%, 100%, one hour each) and embedded in Technovit 8100
17 (Kulzerand Co., Germany) using 4ºC cold polymerization. Sections of 4µm thickness
18 were obtained with a Leica RM2155 rotatory microtome, mounted on 2% (3-
19 Aminopropyl) triethoxysilane (APTEX, Sigma- Aldrich) coated slides, and dried on a
20 slide warmer. Some sections were preserved for immunolocalization, and others were
21 stained with 0.25% toluidine blue for general structure (Feder and O´Brien, 1968), and
22 periodic acid-Schiffs reagent (PAS) for insoluble polysaccharides (Feder and O´Brien, Losada JM, Herrero M, Hormaza JI and Friedman WE 9
1 1968). Light micrographs were obtained with a Zeiss Axio Imager A2 microscope with
2 an Axio Vision HRC camera and AxioVision4.8 digital software.
3 AGP detection in Magnolia stigmas — To avoid possible AGP leakage, stigmatic whole
4 mounts were stained with beta-glucosyl-Yariv-reagent (β-GlcYR) (Biosupplies,
5 Australia) (Popper, 2011) at three flower developmental stages. This dye precipitates
6 AGPs in situ producing a red color product (Yariv et al., 1967). Whole mount β-GlcYR
7 precipitations were recorded with a Zeiss Discovery V12 stereomicroscope. Whole
8 mounts and sections were also used to immunolocalize two AGP epitopes with the JIM8
9 monoclonal antibody, which recognizes an unknown epitope (Pennell et al., 1991) and
10 with JIM13 monoclonal antibody which tags the epitope βGlcA3αGalA12Rha
11 (Knox et al., 1991) (Carbosource services, University of Georgia, USA). Samples were
12 soaked in PBS, pH 7.3 for 5 min, preincubated with 5% bovine serum albumin (BSA) in
13 PBS for 5 min, and then incubated at room temperature for 1 h with JIM8 and JIM13
14 primary monoclonal antibodies, followed by three washes in PBS for five min each.
15 Samples were then incubated with a 1/25 dilution of the Alexa 488 fluorescein
16 isothiocyanate (FITC)-conjugated with the anti-rat IgG secondary antibody (F-1763;
17 Sigma) in 1% BSA in PBS for 45 min in the dark, followed by three washes in 1X PBS
18 for 15 min (Solís et al., 2008). Controls were performed for each sample, by incubating
19 with 1% BSA instead of the primary antibody. To reveal the presence of the green
20 fluorescent FITC signal in relation to cell walls, samples were counterstained with
21 calcofluor white for cellulose and other polysaccharides (Hughes and McCully, 1975).
22 RESULTS Losada JM, Herrero M, Hormaza JI and Friedman WE 10
1 Final stages of flower development and stigma maturation in Magnolia virginiana —M.
2 virginiana showed asynchronous flowering within an individual tree and, thus,
3 geitonogamy is possible although individual flowers are protogynous. Following flower
4 bud development (Figs. 1A-B), individual flowers open twice on two consecutive days
5 (Video S1). On the first day of flower opening, creamy white tepals opened at dusk
6 (19:00h) exposing the stigmas while anthers were not yet dehiscent (Fig. 1C). Three to four
7 hours later, the inner tepals folded inward and formed a chamber that enclosed the stigmas
8 (Fig. 1D). The tepals reopened late in the afternoon of the following day (18:00h) when
9 detached dehisced anthers presented pollen on the reflexed tepals (Fig. 1E).
10 In flower buds just before opening on the first day, conduplicate carpels showed a long
11 domed stigmatic surface with bilateral symmetry (Fig. 1F). Stigmas curled prior to first
12 flower opening as multiple papillae elongated, forming two stigmatic crests along the
13 suture line (Fig. 1G). Concomitant with stigmatic coiling, wet papillae acquired maximum
14 turgidity on the first day of flower opening in the female phase (Fig. 1H). Flower
15 (re)opening on the second day was characterized by brown stigmas (Fig. 1I), suggesting
16 stigma degeneration.
17 To evaluate the possible contribution of AGPs to stigmatic secretion, AGPs were stained
18 with βGlc-YR (beta-glucosyl-Yariv-reagent), a dye that forms a red colored precipitate
19 with AGPs, and was evident at flower bud stages prior to flower opening (Fig. 2A). At
20 these stages, labelling with JIM13 monoclonal antibodies showed a discrete AGP presence
21 on the stigmatic surface (Fig. 2B), but no fluorescent signal was observed on the stigmas
22 after immunolabelling with JIM8 monoclonal antibodies (Fig. 2C). One day before flower
23 opening, an intense red color on the stigma surface indicated AGP precipitation with Yariv Losada JM, Herrero M, Hormaza JI and Friedman WE 11
1 reagent (Fig. 2D). While epitopes recognized by JIM13 monoclonal antibodies were
2 profusely present in the secretions between papillae (Fig. 2E), those recognized by JIM8
3 monoclonal antibodies were not detected on the papillae surfaces (Fig. 2F). On the first
4 day of flower opening at the female stage, the wet stigmatic surface stained intensely with
5 βGlc-YR, indicating the presence of AGPs (Fig. 2G). Although epitopes recognized by
6 JIM13 monoclonal antibodies slightly reacted in the stigmatic secretion (Fig. 2H), JIM8
7 monoclonal antibodies detected epitopes at the papillae tips (Fig. 2I).
8 Stigma receptivity and secretions —Hand pollinations were performed at different times
9 during flower development, but no adhered pollen grains were observed on stigmas of the
10 different flower bud stages (Fig. 3A), or one day prior to flower opening (Fig. 3B). While
11 cutin was not detected at the stigma surface, lipid droplets accumulated on the papillae
12 surface, suggesting a `naked´ stigma covered by lipid-rich secretions. Stigmatic receptivity
13 was acquired hours before the first flower opening, as shown by massive pollen grain
14 germination on stigmas (Fig. 3C). Pollen grains stained intensely for lipids, but the stigma
15 surface lacked a cutin layer. Interestingly, following tepal closure on the first day of flower
16 opening, stigmas were no longer able to sustain pollen germination (Fig. 3D). This
17 occurred only five hours after flower opening, indicating a coordination between floral
18 movements and receptivity and a limited window of stigma receptivity in M. virginiana.
19 To investigate possible changes in the stigmas associated with successful pollination,
20 unpollinated and pollinated stigmas were compared (Fig. 4). On the first day of flower
21 opening, at the functionally female stage, stigmas had clear bilateral symmetry, with two
22 papillate stigmatic crests bordering an adaxial narrow suture line of interlocking stigmatic
23 papillae at the outer marginal edge, and squared epidermal cells at the inner marginal edge Losada JM, Herrero M, Hormaza JI and Friedman WE 12
1 (Fig. 4A). Following pollination, papillae lost turgidity as the suture line began to separate
2 (Fig. 4B). A closer view of the top and bottom of the suture line at flower opening showed
3 that the thick cell walls were composed of polysaccharides (Fig. 4C) and that starch
4 accumulated in the inner cells near the suture line (Fig. 4D). Following pollination,
5 enlargement of the suture line was concurrent with starch hydrolysis from the surrounding
6 tissue and the secretion of insoluble polysaccharides (Fig. 4E, F). Unpollinated stigmas at
7 the closed chamber stage at the end of the first day of flower opening showed collapsed
8 papillae, but polysaccharides were profusely present at their surface (Fig. 4G), and starch
9 was further hydrolyzed in the parenchymatic tissue at the bottom of the suture line (Fig.
10 4H).
11 AGPs during pollination —While epitopes recognized by JIM13 monoclonal antibodies
12 were observed on the stigma surface at first flower opening, and JIM8 epitopes were
13 detected on the stigma at pollen reception, the relationship of JIM8 epitopes with pollen
14 grains and pollen tubes was studied by sequential immunodetection in stigmatic whole
15 mounts. A correlative presence of JIM8 epitopes was observed in contact with the exine of
16 germinating pollen grains two hours after pollination (Fig. 5A; video S2), as the generative
17 cell and tube (vegetative) nucleus separated during hydration and initial germination.
18 Concurrent with migration of the vegetative nucleus and the generative cell into the
19 growing pollen tube, JIM8 epitopes were detected in contact with the pollen tube surface
20 (Fig. 5B). Twelve hours after pollination, only residual JIM8 epitopes were detected on the
21 stigmatic surface of M. virginiana (Fig. 5C).
22 To confirm the presence of JIM8 epitopes on the stigma, cross sections were
23 immunostained. JIM8 epitopes were present in the papillae cell walls prior to pollen arrival Losada JM, Herrero M, Hormaza JI and Friedman WE 13
1 (Fig. 6A). While the region of the suture line lacked these epitopes, cells surrounding it
2 labelled for JIM8 epitopes (Fig. 6B). In particular, starch granules in the parenchymatic
3 tissue were coated by these epitopes detected by JIM8 monoclonal antibodies. Twelve
4 hours after pollination, residual JIM8 epitopes were barely detectable in the external
5 papillae cell walls (Fig. 6C). A lack of those epitopes was further noted within the suture
6 line space and the surrounding tissue (Fig. 6D). In contrast, in unpollinated stigmas at the
7 closed chamber stage, JIM8 epitopes were still present on the papillae cell wall surface
8 (Fig. 6E), as well as along the suture line (Fig 6F).
9 DISCUSSION
10 Flower opening and closure movements during the dichogamous cycle of M. virginiana,
11 correlate with the secretion of saccharide-rich molecules to the stigma surface during the
12 female phase. Their brief persistence following pollen germination and tube growth defines
13 a tight window of stigmatic receptivity and points to AGPs as candidates associated with
14 flower receptivity, both in this member of the magnoliids, as well as more generally among
15 flowering plants (Fig. 7).
16 Stigma secretions and protogyny —, Under the environmental conditions where the trees
17 used in this experiment are grown, M. virginiana flowers follow a protogynous cycle over
18 the course of two days, first opening in the female stage for only four to five hours during
19 the evening of the first day of the cycle, then passing through a closed chamber stage which
20 lasts for 19 hours, and finally reopening in the male stage on the evening of the following
21 day (see also Thien, 1974). Protogyny has been reported as a common mechanism of
22 dichogamy in most basal angiosperm lineages with hermaphroditic flowers (Endress, 1990, Losada JM, Herrero M, Hormaza JI and Friedman WE 14
1 2010, 2011). Usually, in species of the genus Magnolia the female phase of the
2 dichogamous cycle is accompanied by an intense release of scents and thermogenesis at
3 tepal closure (Dieringer et al., 1999; Bernhardt, 2000; Seymour et al., 2010; Kameneva and
4 Koksheeva, 2013; Wang et al., 2013). This suite of flowering characteristics has been
5 suggested to be the result of a tight pollinator-flower coevolutionary history (Thien, 1974;
6 Thien et al., 2009; Gottsberger et al., 2012; Wang et al, 2013; Pagnusat and Saunders,
7 2014).
8 Most early-divergent angiosperm lineages, such as Nymphaeales, Austrobaileyales, and
9 some Magnoliales (Igersheim and Endress, 1997; Endress and Igersheim, 2000), have
10 extracellular gel-like secretions on the stigmatic surface. Our observations clearly show a
11 sticky surface, which covered the stigma and the suture line where pollen tubes grow, and
12 was briefly present on the stigmas at the time of first flower opening in Magnolia
13 virginiana. As is common in other species with wet stigmas, this exudate is carbohydrate-
14 rich, and its appearance was immediately preceded by massive hydrolysis of transient
15 starch in the stigma of M. virginiana. Several mutually non-exclusive functions have been
16 proposed for the presence of sugars in and on the stigmas. These include providing a
17 medium for pollen germination (Herrero and Dickinson, 1979; Rodrigo et al., 2009; Lora
18 et al., 2010; Nepi et al., 2012), serving as a pollinator reward (Amstrong and Irvine, 1990;
19 Thien et al., 2009; Erbar and Leins, 2013), and possibly aiding in desiccation avoidance
20 (Feild et al., 2009; Nepi et al., 2012). Interestingly, in protogynous flowers nectar is mainly
21 limited to the female phase (for a review, see Erbar 2014).
22 Secretion of AGPs and stigmatic receptivity — Our studies reveal a developmentally
23 regulated secretion of two AGPs labeled for different epitopes (JIM8 and JIM13) in the Losada JM, Herrero M, Hormaza JI and Friedman WE 15
1 stigmas of M. virginiana. Interestingly, the secretory timing for these two AGPs is slightly
2 asynchronous: AGPs labeled with JIM13 appear to be secreted onto the stigmatic surface
3 before flower opening, whereas AGPs labeled with JIM8 are secreted following flower
4 opening and during the initial progamic phase of this species. Their synchronous presence
5 in mature stigmas and their absence after stigmatic receptivity prompts interpretation of
6 their role as markers for pollen receptivity in Magnolia virginiana. Studies on the
7 biochemical properties of extracellular AGPs strongly suggest their involvement in
8 “gelatinization” of cell wall secretory products, thus creating adhesive properties (Seifert
9 and Roberts, 2007; Lora et al., 2011; Moore et al., 2013). Even though information about
10 the role of AGPs during the receptive phase of most angiosperm taxa is spotty, previous
11 work showed a similar pattern in flowers of Malus (Losada and Herrero, 2012, 2014),
12 pointing to a role of JIM13 epitopes in providing an adequate viscous environment for
13 pollen adhesion on the stigmatic surface.
14 Our results also show JIM8 AGP epitopes on the surface of starch grains in the cells
15 flanking the suture line of the carpel in M. virginiana. Co-localization of AGPs with starch
16 grains has been recently reported in maternal seed storage tissues of the basal angiosperm
17 and waterlily, Trithuria (Costa et al., 2013). Different small proteins have been reported
18 on the surface of starch grains in other taxa (Baldwin, 2001), and their biological activity
19 has been linked to starch biosynthesis and/or degradation, but so far AGPs have not been
20 tied to this function (Martin and Smith, 1995). In M. virginiana, starch hydrolysis in the
21 stigmatic cells adjacent to the suture line of stigmas occurs concurrent with pollination.
22 Also concurrently, AGPs were no longer detected in these stigmatic regions, but appeared
23 at the secretion localized in the suture line of unpollinated stigmas, suggesting a coupled Losada JM, Herrero M, Hormaza JI and Friedman WE 16
1 translocation of sugars and JIM8 labeled AGPs towards the pollen tube passage area. It
2 will be interesting to evaluate the mechanism leading to co-localization of AGPs and
3 saccharides at the initial stages of male gametophyte-female sporophyte communication.
4 The possible role of AGPs epitopes in pollen tube growth is further reinforced by the fact
5 that they could no longer be detected in both the suture line and the tissues surrounding it
6 following pollen tube passage, while in unpollinated stigmas they were still present in the
7 extracellular space of the suture line. While additional functional data are needed, a similar
8 spatial and developmental pattern has been reported for the TTS AGP-like protein in
9 Nicotiana transmitting tissue within the style (Cheung et al., 1995; Wu et al., 2000;
10 Twomey et al., 2013) and AGPs have also been localized in the stigmas and transmitting
11 tissues of the style of a number of eudicots (Wu et al., 2000; 2001; Hancok et al., 2005;
12 Coimbra et al., 2007; Suarez et al., 2012). Our controlled pollination experiments show
13 that stigmatic receptivity in M. virginiana lasts for few hours and correlates with the spatio-
14 temporal secretion of two AGP epitopes onto the stigma, thus suggesting that they could
15 mark flower receptivity in this species.
16 AGPs in the pistils of early-divergent angiosperms and implications for pollen-pistil
17 interaction — From an evolutionary perspective, the presence of AGPs in the stigma of
18 Trithuria (Nymphaeales) (Prychrid et al., 2011), and in the extracellular matrix of styles
19 of other basal angiosperms such as Amborella (Amborellales), Trimenia and Kadsura
20 (Austrobaileyales) (Lyew et al., 2007; Sage et al., 2009) and Sarcandra and Chloranthus
21 (Chloranthales) (Hristova et al., 2005) suggest an ancient role of these compounds in the
22 the reproductive process in angiosperms. Results herein are the first to report the presence
23 of AGPs in the maternal sporophytic tissues of a member of the magnoliids, a relatively Losada JM, Herrero M, Hormaza JI and Friedman WE 17
1 ancient clade of flowering plants that originated after the basal angiosperm grade clades
2 (Amborella, Nymphaeales, Austrobaileyales) were established, and may predate the
3 origin of monocots and eudicots (APG III, 2009; Friis et al., 2006; 2011; Magallon et al.,
4 2013; Davis et al., 2014; Maia et al., 2014; Ruhfel et al., 2014). While additional
5 evaluation of other magnoliid taxa would be desirable, our observations in M. virginiana
6 point to a well-coordinated and tight relationship between female receptivity, pollen tube
7 growth, and glycoprotein secretions within the gynoecial tissues of the stigma and style
8 that appears to be general among the major clades of extant flowering plants.
9
10
11 FIGURE LEGENDS
12 Fig. 1. Magnolia virginiana flower and stigma development. (A) Flower, green bud stage,
13 seven days before first opening (-7D). (B) Tepals (white) on the morning of the first day
14 of (but prior to) flower opening (0D). (C) First flower aperture – female phase – the evening
15 of the first day of flower opening. (D) Four-to five hours after initial opening of flower, the
16 inner tepals form a chamber enclosing the stigmas. (E) Flower re-opening the evening of
17 the next day, with dehiscent anthers. (F) Bud stage (tepals removed) showing immature
18 stigmas (arrows) with a domed surface (inset). (G) Hours before first flower opening
19 (tepals removed), anthers are yellow but undehisced, stigmas are curled at the tips (arrows),
20 and stigmatic papillae are prominent (inset). (H) Shortly after first flower opening, stigmas
21 are completely curled (arrows) exposing turgid papillae (inset). (I) Shortly after flower re-
22 opening for male phase, stigmas (arrows) are brownish (inset). Anthers are not visible in Losada JM, Herrero M, Hormaza JI and Friedman WE 18
1 this image. Field photographs of Magnolia flowers (A-E), and gynoecia development under
2 the stereo microscope. (F-I) scale bars: 1000µm, insets 250µm.
3
4 Fig. 2. Developmental expression of AGPs in stigmas of Magnolia virginiana. (A) Light
5 AGP precipitation (red color, arrowhead) on the stigma surface at the flower bud stage. (B)
6 Weak JIM13 epitope labelling (arrowhead) on the stigma surface at the flower bud stage.
7 (C) AGPs detected by JIM8 monoclonal antibodies were not present on the stigma at the
8 flower bud stage (D) Massive AGP precipitation (red color, arrowheads) on the stigma
9 surface one day before female flower opening. (E) AGPs labelled with JIM13 were
10 abundant between papillae (arrowheads) at this stage. (F) JIM8 labelled AGPs were
11 undetected on the papillae surface at this stage. (G) Jelly-like red stigma after AGP
12 precipitation (red color, arrowheads) at the time of first (female phase) flower opening. (H)
13 Weak detection of AGPs labelled with JIM13 on the the stigma surface. (I) JIM8 labelled
14 AGPs detected at the papillae tips (arrowheads). Whole mounts of stigmas stained with
15 βGlcYR for AGPs precipitation (A, D, G). Maximum projection of 3D confocal z-stacks
16 of stigmas; AGPs recognized with JIM13 (B,E,H), and JIM8 (C,F,I) monoclonal
17 antibodies, shown with a secondary Alexa488 antibody linked to FITC (fluorescent green),
18 and counterstained with calcofluor white for cellulose (grey) (E,F,H,I); scale bars: 500 µm.
19
20 Fig. 3. Stigmatic receptivity and lipids on the stigma of Magnolia virginiana. (A) No pollen
21 grains were observed on the immature stigma surface after hand pollination. Lipids only
22 stained underneath stigmatic papillae (arrowheads; nuclei fluoresce blue). (B) Pollen grains
23 were unable to adhere on the stigma surface one day before flower opening, and lipid Losada JM, Herrero M, Hormaza JI and Friedman WE 19
1 stained droplets (yellow, arrowheads) could be detected on the papillae surface. (C)
2 Massive pollen grain adhesion following hand pollination the morning of the first day of
3 flower opening, and pollen tube germination (arrows; lipids on pollen grains bright
4 yellow). The stigma lacked a cutin layer. (D) Only four hours after flower opening, stigmas
5 lost the ability to hydrate and the germination of pollen grains did not occur (arrows).
6 Maximum projections of z-stack confocal images of pollen supplemented stigmas stained
7 with auramine O for lipids (yellow) and counterstained with DAPI for nuclei (blue) (A).
8 Scale bars: 100µm.
9
10 Fig. 4. Ontogenetic changes in Magnolia virginiana stigmas with pollination. (A)
11 Transverse section of stigma showing two symmetric papillae columns and a suture line
12 formed by a narrow space left by epidermal cells at female flower opening. Black
13 rectangles denote two areas detailed in the images below: the outer (C,E,G) and inner
14 (D,F,H) marginal edge of the suture line. (B) Papillae collapse after pollination and the
15 suture line enlarges. (C) Papillae with thick cell walls at female flower opening. (D) Small
16 starch grains accumulated in the epidermal layer surrounding the suture line (dotted line),
17 while large starch grains (arrows) were present in the subdermal ground tissues. (E)
18 Following pollination, polysaccharide secretion was abundant on the upper papillae and at
19 the suture line. (F) Following pollination, the enlarged suture line was filled with
20 polysaccharides as starch vanished from the epidermal cells bordering the suture line
21 (dotted line) as well as the subdermal ground tissues proximal to the suture line. (G)
22 Unpollinated stigma showing degenerated papillae. (H) Starch also vanished in
23 unpollinated stigmas, while modest levels of polysaccharide secretions remained in the Losada JM, Herrero M, Hormaza JI and Friedman WE 20
1 epidermal layer surrounding the suture line (dotted line). Technovit 8100 embedded 4 µm
2 cross-sections of Magnolia virginiana stigmas stained with toluidine blue for general
3 structure (A, B), and periodic acid shiffs –PAS reagent for insoluble polysaccharides (C-
4 H) at female flower opening (A,C,D), twelve hours after pollination (B,E,F), and
5 unpollinated stigmas twelve hours after female opening (G,H). P, papilla; pg, pollen grain.
6 Scale bars: 50µm.
7
8 Fig. 5. Sequential detection of JIM8 epitopes during pollen germination in Magnolia
9 virginiana. (A) JIM8 labelled AGPs (arrowheads) in contact with pollen grain exine
10 (arrow). Generative (asterisk) and vegetative nuclei were still inside the pollen grain, two
11 hours after pollination. (B) JIM8 labelled AGPs (arrowheads) in contact with elongating
12 pollen tubes (arrows) as nuclei (asterisk) migrated into the pollen tube four hours after
13 pollination. (C) A residue of JIM8 labelled AGPs were detected in the collapsed stigmatic
14 papillae twelve hours after pollination. 3D confocal reconstructions hand pollinated
15 stigmas immunolocalized for JIM8 epitopes (fluorescent green), counterstained with DAPI
16 for nuclei (blue). pg, pollen grain. Scale bars: 10µm.
17
18 Fig. 6. Localization of AGPs labelled with JIM8 epitopes in cross sections of stigmas of
19 Magnolia virginiana. (A) AGPs labelled with JIM8 (arrowheads) localized across papillae
20 cell walls at the female flower opening stage. (B) AGPs labelled with JIM8 associated with
21 starch grains (arrowheads) of the subdermal tissue proximal to the epidermis that defines
22 the suture line (dotted line). (C) Twelve hours after pollination a residuum of JIM8 labelled
23 AGPs were detected at the papillae cell walls. (D) The suture line lacked JIM8 labelled Losada JM, Herrero M, Hormaza JI and Friedman WE 21
1 AGPs both in the tissue around and within the suture. (E) In unpollinated stigmas, JIM8
2 labelled AGPs (arrowheads) were evident on the external side of the papillae (arrowheads).
3 (F) JIM8 labelled AGPs (arrowheads) were abundant in the suture line and epidermal cells
4 that define the suture, but no longer present in the subdermal tissues proximal to the suture
5 line in unpollinated stigmas. 4µm sections of Technovit 8100 embedded Magnolia
6 virginiana stigmas at female flower opening (A,B), twelve hours after pollination (C,D),
7 and unpollinated stigmas twelve hours after flower opening (E,F) showing
8 immunolocalization of JIM8 epitopes in fluorescent green, and counterstained with
9 calcofluor white for cell walls (grey). P, papilla; pg, pollen grain. Scale bars: 20µm.
10
11 Fig. 7. Temporal scheme of the flowering cycle in Magnolia virginiana in transverse
12 sectional views of the stigma during the period of flower receptivity. Secretion of AGPs
13 recognized by JIM13 monoclonal antibodies on the stigmatic surface (green) is
14 concomitant with starch accumulated in the parenchyma of the stigma surrounding the
15 suture line (red circles). Starch appears to be surrounded by AGPs recognized by JIM8
16 monoclonal antibodies (green), and both are secreted to the extracellular space of the suture
17 line during pollen tube elongation (purple), disappearing soon thereafter.
18
19 SUPPORTING INFORMATION
20 Video S1: Time lapse video of floral movements associated with Magnolia virginiana
21 protogynous flowering cycle.
22 Video S2: Projection of Z-axis images of wholemount of Magnolia virginiana pollen grain
23 two hours after pollination. Losada JM, Herrero M, Hormaza JI and Friedman WE 22
1
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