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. 2015 Jun 22;116(3):403–411. doi: 10.1093/aob/mcv079

Dynamic distribution and the role of abscisic acid during seed development of a lady’s slipper orchid, Cypripedium formosanum

Yung-I Lee 1,2,*, Mei-Chu Chung 3, Edward C Yeung 4, Nean Lee 5
PMCID: PMC4549955  PMID: 26105185

Abstract

Background and Aims Although abscisic acid (ABA) is commonly recognized as a primary cause of seed dormancy, there is a lack of information on the role of ABA during orchid seed development. In order to address this issue, the localization and quantification of ABA were determined in developing seeds of Cypripedium formosanum.

Methods The endogenous ABA profile of seeds was measured by enzyme-linked immunosorbent assay (ELISA). Temporal and spatial distributions of ABA in developing seeds were visualized by immunohistochemical staining with monoclonal ABA antibodies. Fluoridone was applied to test the causal relationship between ABA content and seed germinability.

Key Results ABA content was low at the proembryo stage, then increased rapidly from 120 to 150 days after pollination (DAP), accompanied by a progressive decrease in water content and seed germination. Immunofluorescence signals indicated an increase in fluorescence over time from the proembryo stage to seed maturation. From immunogold labelling, gold particles could be seen within the cytoplasm of embryo-proper cells during the early stages of seed development. As seeds approached maturity, increased localization of gold particles was observed in the periplasmic space, the plasmalemma between embryo-proper cells, the surface wall of the embryo proper, and the inner walls of inner seed-coat cells. At maturity, gold particles were found mainly in the apoplast, such as the surface wall of the embryo proper, and the shrivelled inner and outer seed coats. Injection of fluoridone into capsules resulted in enhanced germination of mature seeds.

Conclusions The results indicate that ABA is the key inhibitor of germination in C. formosanum. The distinct accumulation pattern of ABA suggests that it is synthesized in the cytosol of embryo cells during the early stages of seed development, and then exported to the apoplastic region of the cells for subsequent regulatory processes as seeds approach maturity.

Keywords: Abscisic acid, developing seed, embryo, fluoridone, seed germination, immunolocalization, lady’s slipper orchid, Cypripedium formosanum, Orchidaceae

INTRODUCTION

Embryo development in Orchidaceae is unique, given that at the time of seed dispersal the embryos are arrested at the globular-like stage, with no morphological differentiation into the cotyledon or an embryonic axis. Upon germination, the embryo develops into a protocorm. In a developing protocorm, a shoot meristem develops, and a root subsequently appears at the base of the newly formed shoot. Since Knudson (1922) reported that orchid seeds could germinate asymbiotically on artificial media, many orchid species have been successfully propagated in vitro from seeds. However, seed germination of terrestrial orchids, such as Cypripedium species, has been more difficult than that of tropical epiphytic orchids (Arditti and Ernst, 1993; Rasmussen, 1995). There are three putative reasons for the poor germination of terrestrial orchid seeds: (1) the restriction of water uptake due to the impermeable seed coat (Veyret, 1969); (2) the presence of germination inhibitors in mature seeds (Burgeff, 1936; Linden, 1980; Van Waes and Debergh, 1986); and (3) the onset of dormancy in mature seeds (Veyret, 1969).

Cypripedium formosanum, commonly known as the ‘Taiwan lady’s slipper’, is usually found in the high mountains (around 2000 m above sea level) in Taiwan. Because of its delicate flowers, C. formosanum shows potential for the horticultural trade. However, the plants currently sold by nursery growers are mainly collected from wild stands. Hence, there is an urgent need to conserve this endangered species. In a previous study, we documented the key anatomical features in embryo development of C. formosanum in association with the ability of embryos to germinate in vitro (Lee et al., 2005). A better understanding of its reproductive physiology will provide valuable information concerning the propagation of those hard-to-germinate species.

In developing seeds, abscisic acid (ABA) is necessary for inducing the synthesis of reserve proteins and lipids, as well as for the onset of seed dormancy and the acquisition of desiccation tolerance (Marion-Poll, 1997). Mature seeds of Epipactis helleborine, a hard-to-germinate orchid species, contained five times the amount of free ABA compared with the readily germinating Dactylorhiza maculata (Van der Kinderen, 1987). In D. maculata, Van Waes and Debergh (1986) found that there was no detectable free ABA in seeds after a surface sterilization with 5 % Ca(OCl)2 for 2 h. The application of ABA led to inhibited germination in these seeds. These results suggest that ABA is a germinating inhibitor in orchid seeds (Van der Kinderen, 1987). Although a certain amount of information is available, there are no detailed studies about the changes in the level and distribution of endogenous ABA during embryogenesis in orchid seeds.

The development of polyclonal antibodies against ABA has made it possible to reveal the distribution of ABA in plant cells (Sossountzov et al., 1986). Furthermore, commercialized monoclonal hybridoma antibodies raised against an ABA–bovine serum albumin (BSA) conjugate (ABA-15-I-C-5; Agdia-Phytodetek/Linaris, Wertheim-Bettingen, Germany) have been applied to visualize ABA accumulation in xylem parenchyma cells in plant tumours and stems (Wachter et al., 2003), and ABA distribution in maize roots during its radial transport (Schraut et al., 2004). Knowledge about the precise localization of ABA would greatly help in our understanding of its physiological function in seed development. In order to gain a better insight into the role of ABA in orchid seed development, we examined dynamic changes of endogenous ABA content in developing seeds of C. formosanum from the proembryo stage to maturation, and determined the sub-cellular ABA distribution in the embryo proper and seed coat with mouse monoclonal antibodies against ABA using immunolocalization procedures. To test further the causal relationship between ABA and seed germination, the ABA biosynthetic inhibitor fluoridone was injected into the capsule cavity and the germinability of seeds was monitored. Based on our results, the level and sub-cellular distribution of endogenous ABA were coincident with a decrease in water content, a decrease in the capability for germination in vitro, and the progression of embryo maturation. The inhibition of endogenous ABA biosynthesis using fluoridone promoted mature seed germination.

MATERIALS AND METHODS

Plant materials and seeds sampling

Plants of Cypripedium formosanum were cultivated on the Mei-Fong high-land farm (2100 m above sea level), Taiwan. To ensure a good fruit set and seed viability, the flowers were manually self-pollinated in March. In each experiment, the capsules were randomly collected at regular intervals after pollination. Light micrographs showing the embryo at different developmental stages are presented in Supplementary Data Fig. S1. For the assays of endogenous ABA and germination experiments, the same capsules were used. For the measurement of the water content of developing seeds, because of the limited fresh weight of seeds per capsule, several capsules were used per sample.

Measurement of the water content of developing seeds

Three capsules were randomly collected at intervals of 15 d from 60 days after pollination (DAP) to 180 DAP. Seeds at different developmental stages (0·1 g for each stage) were dissected carefully from the placenta and were then dried at 70 °C for 48 h. The water content was estimated as the percentage of water loss: fresh weight minus dry weight relative to fresh weight.

Measurement of the endogenous ABA content

The procedure for immunoassays of endogenous ABA content has been described in detail by Lee et al. (1993). Briefly, for ABA extraction, the seeds from three capsules were dissected carefully from the placenta and frozen immediately in liquid nitrogen, and then stored at −70 °C for further analyses. The seeds at different development stages were homogenized with a mortar and pestle in an extraction solution (80 % methanol, 2 % glacial acetic acid). Extraction was carried out at 4 °C with shaking for 48 h under darkness. An internal standard, 166·5 Bq of dl-[G-3H]ABA (Amersham Biosciences, Buckinghamshire, UK), was added for estimation of the extraction efficiency. Average recovery ranged from 71 to 76 %. Extracts were filtered through filter paper (Whatman No. 1), and then further rinsed twice with extraction solution. The filtrates were dried in vacuo at 30 °C then resuspended in 100 % methanol. A solution of 0·2 m (NH4)2HPO4 was subsequently added and the samples were allowed to stand for 10 min at 4 °C until ammonium salts formed. Pigments and phenolics in the ammonium salts solution were removed by passing them through a PVP column (Mousdale and Knee, 1979). The combined PVP column-filtered solutions were adjusted to pH 3·0 with 1 m acetic acid. The acidified solution was eluted through a C18 cartridge in order to remove polar compounds. ABA trapped in the C18 cartridge was then eluted with 55 % methanol. The ABA solution was dried in vacuo. Samples were resuspended in Tris-buffered saline (50 mm Tris–HCl, 10 mm NaCl, 1 mm MgCl2, 15 mm NaN3, pH 7·5) and stored at −20 °C for enzyme-linked immunosorbent assay (ELISA) analysis. Quantification of ABA was estimated by an indirect ELISA method according to Walker-Simmons (1987). ABA levels are expressed as ng mg–1 fresh mass.

Seed germination in vitro

Developing capsules were collected and processed for germination tests according to Lee et al. (2005). The capsules were surface-sterilized with a 1 % sodium hypochlorite solution for 20 min. After surface-sterilization, the capsules were cut open, and the seeds were scooped out with forceps and placed onto Thomale GD medium (Thomale, 1957), supplemented with 20 g L–1 sucrose, 100 mL L–1 coconut water and solidified with 2·2 g L–1 Phytagel™ (Sigma-Aldrich Co., St. Louis, MO, USA). The pH value was adjusted to 5·7 before autoclaving. The culture tubes were placed in a growth room at 25 ± 2 °C, in constant darkness. Each developmental stage had 20 replicates, which were composed of seeds from three capsules. In each replicate, there were approx. 100 seeds evenly placed on the surface of the medium. The number of germinating seeds was examined under a Zeiss stereomicroscope (×10 magnification) and recorded. Seed germination is recognized as an embryo emerging from the seed coat. Germination was expressed as a percentage of the total number of seeds cultured.

Tissue preparation for immunocytochemistry

Seeds at different developmental stages were quickly dropped into a solution with 2 % (w/v) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC; Sigma-Aldrich Co.) in 0·1 m sodium phosphate buffer (w/v) and incubated at pH 7·2, at 4 °C for at least 24 h for the penetration of the coupling reagent into the seeds. The EDAC pre-fixation cross-links ABA to the cellular protein network and preserves the antigenicity of ABA towards the anti-ABA monoclonal antibody used in this study (Sotta et al., 1985), which has been applied in the routine protocol for sample preparation in ABA immunolocalization experiments (Betrand et al., 1992; Veselov et al., 2003; Wachter et al., 2003; Schraut et al., 2004). After three 15 min buffer rinses, the materials were post-fixed in a solution of 2 % (w/v) paraformaldehyde and 2·5 % (w/v) glutaraldehyde in the same buffer at 4 °C overnight.

Immunofluorescence observation

The post-fixed samples were rinsed with 0·1 m sodium phosphate buffer three times for 15 min each and dehydrated through an acetone series. The dehydrated samples were infiltrated with increasing concentrations of Technoviet 8100 resin (Kulzer & Co., Wehrheim, Germany), and then they were embedded and polymerized according to the procedure of the manufacturer. Sections of 3 µm were prepared with a Reichert-Jung 2040 Autocut rotary microtome and collected on poly-l-lysine-coated slides (Sigma-Aldrich Co.). The procedure for immunofluorescence detection of ABA has been described in detail by Schraut et al. (2004). In brief, the sections were incubated for 1 h at room temperature in PBST (0·15 m NaCl, 0·2 M phosphate buffer at pH 7·2, plus 0·05 % Tween-20) containing 3 % normal goat serum and 0·5 % BSA to block the unspecific binding of antibodies. The primary monoclonal antibody against ABA (PDM 09347; Agdia-Phytodetek, Elkhart, IN, USA), diluted 1:200 in PBST with 0·5 % BSA, was added to each slide (100 µL per slide), a plastic cover slide was applied and then the slides were incubated in a humid chamber overnight at 4 °C in darkness. After incubation, the slides were washed thoroughly with PBST. After washing, 100 µL of the pre-treated secondary antibody was added to each slide for 1 h at 37 °C. Pre-treatment of the secondary antibody (1:200 in PBST) has been suggested by Peng et al. (2006) in order to reduce the background signals. The procedure for pre-treatment of the secondary antibody is as follows. Approximately 0·5 g of fresh seeds was homogenized in a small volume of ice-cold 90 % acetone in liquid nitrogen, and the mixture was stored overnight at 4 °C. After centrifuging at 13 000 g for 5 min, the supernatant was removed and the pellet was resuspended in a small volume of 90 % acetone, and passed through a filter paper. The dried powder was transferred to an Eppendorf tube and stored at 4 °C. Approximately 50 mg of the powder was added to 500 µL of PBS containing 1 % BSA, and 20 µL of secondary antibody [F0257, fluoresecein isothiocyanate (FITC)-conjugated anti-mouse IgG, Sigma-Aldrich Co.]. The mixture was then incubated in the dark for 24 h at room temperature. After addition of 500 µL of PBS to the mixture, the pre-treated secondary antibody was recovered for use by centrifugation at 10 000 g for 5 min. After a further wash with PBST, the sections were stained with 0·01 % toluidine blue O to quench the tissue autofluorescence (Peterson, 1988), and subsequently mounted in an antifade mounting solution (Vector Laboratories, CA, USA). The locations of signals were viewed with a confocal laser scanning microscope [CLSM; Zeiss LSM 510 Meta, Carl Zeiss AG, Jena, Germany, excitation 543, HFT 543, NFT 635, emission (BP) 560–615]. Controls were made to confirm the specificity of the immunological staining procedure, including (1) incubation with pre-immune mouse IgG (Vector Laboratories) and (2) incubation with FITC-conjugated anti-mouse IgG, omitting the first antibody incubation step. Images were converted to a false colour glow mode to improve the contrasts. The experiments were performed 5–8 times with 60–80 tissue sections each.

Immunogold observation

The post-fixed samples were rinsed with 0·1 m sodium phosphate buffer three times for 15 min each and dehydrated through an ethanol series. The dehydrated samples were embedded and polymerized in London Resin White methacrylate resin (London Company, Basingstoke, UK) according to the procedure of the manufacturer. Ultrathin sections were cut with a diamond knife on an ultramicrotome (Ultracut E, Reichert-Jung, Vienna, Austria) and placed on formvar-coated nickel grids. For transmission electron microscopy (TEM) observation, the immunological staining was performed as previously described (Van Aelst and Van Went, 1992) with minor modifications. The sections were incubated in 3 % normal goat serum and 0·5 % BSA in PBST for 10 min at room temperature. Grids were then transferred to another drop of PBST containing the monoclonal antibody (1:200) against ABA (PDM 09347; Agdia-Phytodetek) and 0·5 % BSA for 1 h at room temperature. After washing thoroughly with PBST, the sections were then treated for 20 min at room temperature with goat anti-mouse–colloid gold conjugates (18 nm; RPN422 Auro-Probe EMGAR G18, Amersham), diluted 1:20 in PBST. After a further wash with PBST and distilled water, the sections were counterstained with uranyl acetate, followed by lead citrate. Controls were made to confirm the specificity of the immunological staining procedure, including (1) incubation with pre-immune mouse IgG (Vector Laboratories, Inc.) and (2) incubation with colloidal gold-conjugated anti-mouse IgG, omitting the first antibody incubation step. A Philip CM 100 transmission electron microscope at 80 kV was used for observation.

Effects of treatments with fluridone or ABA on seed germination

To evaluate the effect of endogenous ABA levels on the germination percentage of mature seeds, immature seeds were treated with fluridone {1-methyl-3-phenyl-5-[3-trifluromethyl(phenyl)]-4-(1H)-pyridinone; Alligare, LLC, AL, USA}, an inhibitor of ABA biosynthesis. Approximately 1 ml of filter-sterilized fluridone at 10 µm was injected into developing capsules at 90 DAP. In the control, 1 mL of filter-sterilized deionized water was applied. Seeds were collected from three treated capsules at maturity (240 DAP) to determine the percentage of seed germination in vitro. The endogenous ABA levels were also determined as described above.

To examine the effect of exogenous ABA levels on seed germination in vitro, seeds of different maturity (seeds collected at intervals of 30 d from 60 DAP to 180 DAP) were inoculated on Thomale GD medium in the presence of 0, 0·0003, 0·0037, 0·0378 or 0·3787 µm (±)-ABA (A1049, Sigma-Aldrich Co.). ABA was filter-sterilized and added to the autoclaved basal medium. The culture procedure and conditions were the same as described above.

Statistical analyses

All experiments were established in a completely randomized design and repeated three times. The data were statistically analysed using analysis of variance (ANOVA) followed by Fisher’s protected least significant difference test.

RESULTS

Changes of water content and endogenous ABA levels in developing seeds

The water content of seeds was determined to be approx. 90 % of the fresh weight at 60 DAP (Fig. 1A). At this stage, the capsules were green, and the seeds were milky white and moist and still attached to the placenta. From 90 DAP to 120 DAP, the seeds began to dry and turned yellowish in colour, and a sharp drop in the water content of the seeds was observed. At 150 DAP, the capsules remained green, but the seeds turned dark brown in colour and had detached from the placenta. The water content of mature seeds (240 DAP) was estimated approximately as >10 %.

Fig. 1.

Fig. 1.

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(A) Changes of water content in developing seeds of C. formosanum; (B) changes in levels of ABA in developing seeds of C. formosanum; (C) changes in germination percentage in developing seeds of C. formosanum. Error bars represent the s.e. of three independent experiments including at least three replicates each.

During the early stages of seed development, the ABA contents were maintained at low levels (1·32–1·96 ng mg–1 f. wt) from 60 DAP to 90 DAP (Fig. 1B). After 90 DAP, the contents of ABA increased rapidly. Maximal ABA contents (13·8 ng mg–1 f. wt) in seeds were observed after 180 DAP. An elevated level of ABA continued from 180 DAP to 240 DAP, indicating that the seeds of C. formosanum maintained a high level of ABA for a prolonged period until the capsules split.

Effect of the timing of seed collection on germination

Figure 1C shows the changes of germination percentage in developing seeds. The seeds collected at 60 DAP germinated poorly but subsequently increased to a maximum (51·2 %) at 90 DAP. From 120 DAP to 180 DAP, the germination percentage decreased progressively. As the seeds matured, after 180 DAP, the germination percentage dropped down to zero.

Effects of fluridone treatment on germination percentage and the endogenous ABA levels of mature seeds

To understand if high endogenous ABA levels of mature seeds prevented germination, immature seeds were treated with 10 µm fluridone, an inhibitor of ABA biosynthesis. The fluridone treatment improved the germination of mature seeds in comparison with the control in which no germination was recorded (Fig. 2A). The mature seeds contained a high ABA level (13·55 ng mg–1 f. wt), whereas in the fluridone-treated capsules, the endogenous ABA level of mature seeds decreased to 6·36 ng mg–1 f. wt (Fig. 2B).

Fig. 2.

Fig. 2.

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The effect of fluridone treatment on (A) seed germination in vitro and (B) endogenous ABA levels of C. formosanum. Data were collected at 150 d after culture initiation. Error bars represent the s.e. of three independent experiments including at least three replicates each.

ABA application suppresses seed germination in vitro

In vitro seed germination capability increased and reached a maximum as the seed developed from 60 DAP to 90 DAP, and then dramatically decreased at 120 DAP (Fig. 3). Applying a relatively low concentration of ABA (0·0037 µm) was sufficient to suppress the germination of seeds at 90 DAP. Seeds collected after 150 DAP showed hardly any germination.

Fig. 3.

Fig. 3.

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The effect of exogenous ABA treatment on seed germination in vitro of C. formosanum. Data were collected at 150 d after culture initiation. Error bars represent the s.e. of three independent experiments including at least three replicates each. DAP, days after pollination.

Changes of ABA localization in developing seeds

The immunofluorescent signal of ABA was undetectable at the proembryo stage (Fig. 4A, B). As the embryo reached the globular stage (120 DAP), the immunofluorescent signal of ABA first presented in the outer walls of the embryo proper, while the signals in the walls of the seed coat were extremely weak (Fig. 4C, D). At maturation (240 DAP), a strong immunofluorescent signal of ABA was detected in the outer wall of the embryo proper, as well as the inner and outer seed coats, but was not detected within the cells of the embryo proper (Fig. 4E, F). In the mature seed, both inner and outer seed coats were dehydrated and were compressed into thin layers that enveloped the embryo proper. No immunofluorescent signals of ABA were observed in the negative control following incubation with pre-immune mouse IgG or the omission of primary antibody, (Fig. 4G, H).

Fig. 4.

Fig. 4.

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Immunocytochemical localization of ABA in developing seeds of C. formosanum. (A, C, E and G) Fluorescence; (B, D, F and H) the merged images of fluorescence and differential interference contrast. (A, B) No visible immunolabelling of ABA was present in the proembryo stage (60 DAP). OS, outer seed coat; IS, inner seed coat. Scale bar = 50 µm. (C, D) At the globular stage (120 DAP), immunolabelling of ABA was first detected in the surface wall of the embryo proper (arrow). Very weak signals were observed in the wall of the seed coat (arrowheads). OS, outer seed coat; IS, inner seed coat. Scale bar = 60 µm. (E, F) A longitudinal section through the mature seed (240 DAP), showing strong immunolabelling of ABA in the surface wall of the embryo proper (arrow), as well as the inner and outer seed coats (arrowheads). OS, outer seed coat; IS, inner seed coat. Scale bar = 50 µm. (G, H) A longitudinal section through the mature seed, showing a negative control with the omission of primary antibody. OS, outer seed coat; IS, inner seed coat. Scale bar = 50 µm.

In order to determine the precise localization of ABA within the seed, the immunogold labelling method was used. At the proembryo stage (Fig. 5A, B), a few immunogold particles were observed in the plastid and cell wall of the embryo. In the maturing seeds, storage products, such as protein bodies and lipid bodies, began to accumulate within the cells of the embryo proper, and the inner seed coat began to shrivel (Fig. 5C). The number of immunogold particles apparently increased in the surface wall of the embryo proper and in the shrivelling cells of the inner seed coat (Fig. 5D). In mature seeds, the embryo was dry and enveloped by the shrivelled inner and outer seed coats, and the embryo cells were filled with protein bodies and lipid bodies (Fig. 6A). Consistent with the immunofluorescent results, immunogold particles could be detected primarily at the surface wall of the embryo proper, as well as the compressed inner and outer seed coats (Fig. 6B, C). At maturity, no immunogold particles were observed in the inner embryonic cells (Fig. 6C). In particular, there are a few immunogold particles deposited at the plasma membrane of embryo proper cells at seed maturity (Fig. 6C). The control with the incubation with pre-immune mouse IgG or the omission of primary antibody showed no immunogold particles in any cells (Fig. 6D).

Fig. 5.

Fig. 5.

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Ultrastructure and immunolocalization of ABA in the developing embryos of C. formosanum. (A) Light micrograph showing a longitudinal section through a proembryo. IS, inner seed coat; OS, outer seed coat. Scale bar = 20 µm. (B) In the proembryo, only a few immunogold particle-labelled ABA molecules (arrows) appeared in the plastid and the cell wall of embryo cells. CW, cell wall; P, plastid; V, vacuole. Scale bar = 1 µm. (C) Light micrograph showing a longitudinal section through a maturing globular embryo. At this stage, storage products, such as protein bodies, accumulated within the cells of the embryo proper. The cells of the inner seed coat began to shrivel. IS, inner seed coat; OS, outer seed coat. Scale bar = 40 µm. (D) In the maturing globular embryo, more immunogold particle-labelled ABA molecules (arrows) were localized in the surface wall (SW) of the embryo proper and the cells of the compressing inner seed coat (IS). Scale bar = 1 µm.

Fig. 6.

Fig. 6.

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Ultrastructure and immunolocalization of ABA in the mature seed of C. formosanum. (A) Light micrograph showing a longitudinal section through the mature seed. While the seed turned desiccated and brown at maturity, the embryo proper was enveloped by the shrivelled seed coats. IS, inner seed coat; OS, outer seed coat. Scale bar = 20 µm. (B) At maturity, the outer seed coat became dehydrated and compressed into a thin layer. A few immunogold particle-labelled ABA molecules (arrows) were found in the dehydrated outer seed coat (OS). Scale bar = 1 µm. (C) In the mature seed, immunogold particle-labelled ABA molecules (arrows) mainly accumulated in the surface wall (SW) of the embryo proper and the shrivelled inner seed coat (IS). Scale bar = 1 µm. (D) The control staining. Thin sections were incubated with the mouse pre-immune IgG instead of anti-ABA antibody. No immunogold particles were observed in these treatments. IS, inner seed coat; L, lipid body; SW, surface wall. Scale bar = 1 µm.

DISCUSSION

ABA and orchid seed development

Abscisic acid has been shown to play an important role in regulating different aspects of embryo development and seed germination (Bewley and Black, 1985). In this study, as shown in Fig. 1, the ABA content in C. formosanum developing seeds continued to increase rapidly from 60 to 150 DAP, and would reach a maximum level as the seed approaches maturation (180 DAP), maintaining such a high level until the capsule split (Fig. 1B). A similar observation was reported in mature seeds of Calanthe tricarinata, a cool-climate terrestrial orchid (Lee et al., 2007). The high ABA level may ensure seed germination is delayed. In C. formosanum, since seed dispersal usually occurs during the late autumn in high mountains of Taiwan, preventing seeds from germinating right away in the severe cold environment is a suitable germination strategy. The profile of ABA levels observed in our studies is different from those observed in other species, such as cereals (Goldbach and Michael, 1976; King, 1976; Kawakami et al., 1997) and tomato (Hocher et al., 1991), in which maximum ABA levels were found at the beginning of maturation followed by a decline. The lower ABA levels as seed matured ensure relative ease of germination.

In orchids, the distinct profile of ABA levels may also relate to the unique pattern of embryo development. It has been shown in arabidopsis that ABA is involved in the induction of storage protein accumulation and the regulation of seed dormancy (Karssen et al., 1983; Rivin and Grudt, 1991; McCarty, 1995; Kagaya et al., 2005). The structure of orchid seed is quite simple, with a globular-like embryo housed within a thin seed coat and the absence of an endosperm. The lack of an endosperm may have slowed the process of storage product synthesis and accumulation. The high levels of ABA observed can promote storage product synthesis and deposition, ensuring sufficient food reserves are present within the embryo for seed germination.

ABA as the key inhibitor of seed germination

A relationship between increased ABA levels and low germination capability was found in C. formosanum. In this study, a maximum germination percentage was obtained at 90 DAP, while the ABA content was still low (1·96 ng mg–1 f. wt). At 120 DAP, a 2-fold increase in ABA level (4·08 ng mg–1 f. wt), although still small, dramatically suppressed seed germination (Fig. 1B, C). The application of a very small amount of exogenous ABA at 0·0037 µm was sufficient to inhibit the germination of immature seeds (Fig. 3). These results indicated that orchid seed germination is highly sensitive to a small increase in ABA level. It is well established in the literature that tissues at different developmental stages differ in their sensitivity to ABA. The difference in sensitivity to ABA is crucial in the control of seed dormancy (Walker-Simmons, 1987; Bianco et al., 1994; Wang et al., 1995; Grappin et al., 2000). The sensitivity towards ABA may have contributed to the observed unique embryo developmental pattern in orchids, i.e. having a rudimentary embryo with no distinct tissue formation. A small increase in ABA may be sufficient to suppress/terminate the histodifferentiation and mitotic cell divisions that caused dormancy. As the seeds approached maturity (after 150 DAP), the accumulation of high ABA levels seriously suppressed the seed germination.

According to Van der Kinderen (1987), ABA was relatively high in the hard-to-germinate orchid species, E. helleborine, which led to the hypothesis that ABA might inhibit seed germination of temperate terrestrial orchids. In cacao zygotic embryos, the endogenous ABA contents correspond well to their capacity to germinate in vitro (Pence, 1992). In a previous report, cultured rapeseed embryos showed a strong positive correlation between the endogenous ABA contents detected in the excised developing embryo and at the lag period before the onset of embryo axial extension (Finkelstein and Crouch, 1986). In order to demonstrate a causal relationship between ABA levels and seed germination capability, a fluridone treatment was conducted. The application of fluridone to developing seeds or mature seeds has been shown to cause vivipary or alleviate dormancy by removing the effects of endogenous ABA (Fong et al., 1983; Yoshioka et al., 1998; Ali-Rachedi et al., 2004; Kusumoto et al., 2006). In the present study, the application of fluridone to developing seeds increased the germination percentage of mature seeds, and the endogenous ABA level of mature seeds was also reduced by fluridone application (Fig. 2). This experimental approach clearly indicates that ABA is an important determinant of germination in temperate terrestrial orchids.

Immunological staining of ABA in developing seeds

The temporal and spatial changes in the distributions of phytohormones provide important clues as to their specific roles in regulating physiological processes. Several studies have been performed in an attempt to localize the position of phytohormones in plant tissues (Zavala and Brandon, 1983; Sotta et al., 1985; Sossountzov et al., 1986; Eberle et al., 1987; Ohmiya et al., 1990). At present, little information is available about the compartmentation of ABA in seeds and how it changes during development (Welbaum et al., 2000). Our data provide direct evidence for the first time of ABA distribution in the embryo cells of developing orchid seeds. Immunofluorescent signals of ABA observed by confocal laser scanning microscopy revealed that the intensity of immunofluorescent signals became stronger as the seeds approached maturity, and were predominantly localized at the surface wall of the embryo proper, as well as the inner and outer seed coats (Fig. 4). The higher intensity of the immunofluorescent signal reflected the increment of endogenous ABA levels as the seeds approached maturity. The precise localization of ABA within developing seeds was further examined by immunoelectron microscopy. In the proembryo stage, immunogold particles were observed in the plastid and cell wall of embryo cells (Fig. 5B), indicating that the embryo cells are capable of synthesizing ABA. As the seeds matured, immunogold particles were primarily found in the surface wall of the embryo proper, as well as the compressed inner and outer seed coats (Fig. 6C). ABA accumulated in the apoplast can have a functional role to play. In the study of sensing, signalling and transport of ABA in guard cells, it has been proposed that ABA is exported from the cells in which it is biosynthesized to the apoplast, and then transported from the apoplast into guard cells to facilitate stomatal closure (Umezawa et al., 2010). In C. formosanum, the distinct accumulation pattern of ABA may suggest that ABA is synthesized in the cytosol of orchid embryo cells during the early stage of seed development, and then exported to the apoplastic region of embryo cells for subsequent regulatory processes as the seeds approach maturity and subsequent germination events.

In conclusion, ABA is confirmed as a regulatory factor in embryo development and seed germination of C. formosanum. Reducing the endogenous ABA level of mature seeds by fluridone application improved the germination percentage. Our finding of preferential accumulation of ABA in the apoplastic region of embryo cells as the seeds approach maturity suggests that apoplastic ABA plays a role in the physiological modulation of dormancy. Therefore, the accumulation of a high ABA level in mature seeds may help seeds maintain their dormancy status to escape from the harsh environment.

SUPPLEMENTARY DATA

Supplementary data are available online at www.aob.oxfordjournals.org and consist of Figure S1: light micrographs showing the embryo of C. formosanum at different stages.

Supplementary Data
supp_116_3_403__index.html (849B, html)

ACKNOWLEDGEMENTS

This work was supported by grants from the National Science Council, Taiwan, ROC [NSC 97-2313-B-178-001] and National Museum of Natural Science, Taiwan, ROC to Y.-I.L., from Academia Sinica, Taiwan to M.-C.C., from the Natural Sciences and Engineering Research Council of Canada to E.C.Y., and from the Council of Agriculture of Taiwan to N.L. We also thank Professor Ching-Huei Kao and Professor Huu-Sheng Lur of the Department of Agronomy, National Taiwan University, for their advice and help with the endogenous ABA analysis. We also thank Dr Wann-Neng Jane and Miss Mei-Jane Fang (Plant Cell Biology Core lab, Institute of Plant and Microbial Biology, Academia Sinica) for the use of a confocal laser-scanning microscope and a transmission electron microscope.

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Associated Data

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Supplementary Materials

Supplementary Data
supp_116_3_403__index.html (849B, html)
supp_mcv079_aob-15022-s01.pdf (151.6KB, pdf)

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