AOBPreview originally published online on September 12, 2005
Annals of Botany 2005 96(6):965-980; doi:10.1093/aob/mci250
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Investigations into Seed Dormancy in Grevillea linearifolia, G. buxifolia and G. sericea: Anatomy and Histochemistry of the Seed Coat
1 Ecology Research Group, University of Western Sydney, Locked Bag 1797, Penrith South DC, NSW 1797, Australia and 2 School of Biological, Earth and Environmental Sciences, University of New South Wales, Sydney, NSW 2052, Australia
* For correspondence. E-mail c.morris{at}uws.edu.au
Received: 29 March 2005 Returned for revision: 23 May 2005 Accepted: 5 July 2005 Published electronically: 12 September 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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• Background and Aims Seeds of east Australian Grevillea species generally recruit post-fire; previous work showed that the seed coat was the controller of dormancy in Grevillea linearifolia. Former studies on seed development in Grevillea have concentrated on embryology, with little information that would allow testing of hypotheses about the breaking of dormancy by fire-related cues. Our aim was to investigate structural and chemical characteristics of the seed coat that may be related to dormancy for three Grevillea species.
• Methods Seeds of Grevillea linearifolia, Grevillea buxifolia and Grevillea sericea were investigated using gross dissection, thin sectioning and histochemical staining. Water movement across the seed coat was tested for by determining the water content of embryos from imbibed and dry seeds of G. sericea. Penetration of intact seeds by Lucifer Yellow was used to test for internal barriers to diffusion of high-molecular-weight compounds.
• Key Results Two integuments were present in the seed coat: an outer testa, with exo-, meso- and endotestal (palisade) layers, and an inner tegmen of unlignified sclerenchyma. A hypostase at the chalazal end was a region of structural difference in the seed coat, and differed slightly among the three species. An internal cuticle was found on each side of the sclerenchyma layer. The embryos of imbibed seeds had a water content six times that of dry seeds. Barriers to diffusion of Lucifer Yellow existed at the exotestal and the endotestal/hypostase layers.
• Conclusions Several potential mechanisms of seed coat dormancy were identified. The embryo appeared to be completely surrounded by outer and inner barriers to diffusion of high-molecular-weight compounds. Phenolic compounds present in the exotesta could interfere with gas exchange. The sclerenchyma layer, together with strengthening in the endotestal and exotestal cells, could act as a mechanical constraint.
Key words: Seed coat structure, Grevillea linearifolia, Grevillea buxifolia, Grevillea sericea, histochemistry, seed dormancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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All Grevillea species from south-east Australia produce a hard, dry seed that is shed from the parent plant after dehiscence of the mature fruit (Olde, 1997
; Auld and Denham, 1999). Seeds from most of these species possess an oil-filled body, the elaiosome (located at the chalazal end of the seed), that is attractive to many ant species and results in the removal of the seed into the nest of large ant species (Auld and Denham, 1999). Hence, many Grevillea seeds are incorporated into the seed bank where they lie dormant until recruitment in an immediate post-fire period (Auld and Tozer, 1995). Laboratory studies of the germination characteristics of these species have shown that fire-related signals such as smoke or heat shock stimulate germination (Edwards and Whelan 1995; Kenny, 2000; Morris, 2000). In addition, scarification of the seed coat increases germination in some Grevillea species (G. barkylana; Edwards and Whelan, 1995), sometimes in combination with heat shock (G. buxifolia) or smoke (G. diffusa, G. mucronulata, G. speciosa; Morris, 2000).
Stimulation of germination by treatments such as heat shock and scarification suggests that the seed coat plays a role in dormancy; this hypothesis was tested for G. linearifolia, a species that shows increased germination in response to smoke and heat shock (Morris, 2000). Removal of the seed coat in G. linearifolia results in germination of all embryos, establishing the seed coat as the primary controller of dormancy in this species (Morris et al., 2000).
Seed coat dormancy is a widespread phenomenon, and there are several mechanisms by which the seed coat can impose dormancy (Bewley and Black, 1994). The seed coat can (1) mechanically restrict germination of the embryo, (2) restrict the exit of germination inhibitors from the embryo, (3) contain germination inhibitors, (4) restrict water uptake and (5) restrict oxygen uptake. Evidence to date allows judgement to be made on some of these possible mechanisms for Grevillea species. The seed coat does not appear to restrict water uptake as unscarified seeds take up water as readily as do scarified seeds (Morris, 2000; Morris et al., 2000). Re-insertion of embryos into dissected seed coats does not inhibit germination of de-coated G. linearifolia (Morris et al., 2000), indicating that germination inhibitors are not released from the seed coat itself.
In view of the importance of the seed coat in the dormancy of these Grevillea species, an improved understanding of the structure of the seed coat is required before hypotheses about how germination treatments affect that structure can be tested (Egerton-Warburton, 1998).
Seed coat structure is described using specific terminologies. The seed coat in all bitegmic species consists of a testa (formed from the outer integument of the ovule) and a tegmen (formed from the inner integument of the ovule) with the outer epidermis of the testa called the exotesta and the inner epidermis called the endotesta; similarly, the outer epidermis of the tegmen is called the exotegmen and inner epidermis the endotegmen. The cell layers between the exo- and endotesta are called the mesophyll, mesotesta or mesotegmen (Corner, 1976). The position of the main mechanical layer within the seed coat determines whether the seed is further described as ‘testal’ or ‘tegmic’. In the Proteaceae the seed coat is considered to be endotestal with a fibrous tegmen. The endotestal cells are thin-walled (with or without lignification) and strengthened by an internal fibrillar reticulum that fills the central cavity. The exo- and endotegmen both consist of narrow, cylindrical fibres with the endotegmal fibres lying across the outer longitudinal fibres (Corner, 1976).
Investigations of seed structure in Grevillea species have been undertaken by Brough (1933), Kausik (1938, 1939) and Venkata Rao (1967). These studies focused on floral development, fertilization and development of the embryo, and the structure of the mature seed was treated very briefly; histochemical techniques were not used. G. robusta was studied by Brough (1933) and Kausik (1938), G. banksii by Kausik (1939), and Venkata Rao (1967) covered a range of species.
Grevillea ovules are orthopterous, hemianatropous or nearly anatropous, bitegmic, crassinucellate, receive a single vascular bundle and are attached laterally to the marginal placenta (Kausik, 1938, 1939; Venkata Rao, 1967). During enlargement of the embryo sac the nucellar tissue may become completely destroyed, or it may persist as a thin layer pressed against the surface of the inner integument (Kausik, 1938, 1939; Corner, 1976). In some species, the nucellus survives as a small group of intact cells at the micropylar and chalazal ends of the ovule. Prior to fertilization, the chalazal nucellar remnant divides to form a distinct region called a ‘hypostase’ that becomes filled with tannins during seed maturation (Kausik, 1938, 1939; Venkata Rao, 1967). In many species producing bitegmic ovules, the cuticles covering the inner epidermis of the outer integument fuse with the cuticle covering the outer epidermis of the inner integument to form a single, thick cuticle; cuticles also occur between the innermost integument and the nucellus and over the surface of the ovule (Maheshwari, 1950).
Olde (1997) divides seeds of Grevillea into three morphological types: (1) the ‘winged’ seed, in which a membranous outgrowth of the testa forms a ‘wing’ that completely surrounds the seed; (2) the ‘oat’-style seed, which lacks the membranous wing but instead has margins that are revolute; and (3) an ellipsoidal/hemispherical seed with a convex outer surface and a flat inner surface.
In Grevillea species the mature seeds are non-endospermic and the embryo completely occupies the embryo sac cavity (Ventaka Rao, 1967). The testa is multilayered (owing to multiplication of the mesophyll cell layers), the cells have lost their tannin and the cells of the inner epidermis although thin-walled are radially elongated like a palisade and contain prominent raphides (Venkata Rao, 1967). The tegmen consists of three crushed cell layers (Venkata Rao, 1967) that in the wing-producing species G. robusta and G. banksii contributes to a fairly hard, protective covering surrounding the embryo (Kausik, 1938, 1939). According to Corner (1976), the cells of the tegmen in Grevillea species are thick-walled, lignified fibres with the middle layer crossing the outer and inner layers of longitudinal fibres.
Most of the studies on seed structure in Grevillea have concentrated on the winged seed-type. However, those east Australian species in which germination responses have been studied (Kenny, 2000; Morris, 2000; Morris et al., 2000) have the ‘oats’-style seed. There is clearly a need for updated information on the basic structure and histochemistry of this style of Grevillea seed to provide a basis for further investigations on the mechanism of action of various germination treatments.
Grevillea linearifolia, G. buxifolia and G. sericea show germination behaviour that is broadly similar, but with some important differences. Almost all seeds of G. buxifolia do not germinate unless treated with fire-related cues in laboratory experiments, whereas a variable fraction of G. sericea and G. linearifolia seeds do germinate without further treatment (Kenny, 2000; Morris, 2000). Heat shock and smoke combined synergistically to increase germination of G. buxifolia, but additively for G. sericea (Kenny, 2000). To understand further the role of the seed coat in imposing dormancy, we have studied the structure of the mature seed of all three species, used histochemical techniques to determine wall composition, and investigated the movement of water and high-molecular-weight compounds in intact seeds. This work is a necessary prerequisite for future experiments to test hypotheses about the breaking of dormancy by fire-related germination cues.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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Seed source
Seeds of G. linearifolia (Cav.) Druce, G. sericea (Smith) R. Br and G. buxifolia (Smith) R. Br. were collected by picking ripe fruits from multiple plants at Narrabeen, New South Wales, in December 2000, returning the fruits to the laboratory and collecting the seeds after the fruits opened. Seeds were stored in paper bags in the laboratory until used (October–December 2001 for gross dissections and initial histochemistry; February–May 2004 for later histochemistry and diffusion experiments).
Gross dissection/photography
Sixteen seeds of each species were placed on moist filter paper in a Petri dish for 24 h, sectioned with a razor blade, and examined for gross morphology, anatomy and colour of pigments in walls and vacuoles.
Tissue preparation for sectioning
Seeds of each species were fixed in a 5 % glutaraldehyde solution in 2·5 % potassium phosphate buffer, pH 6·8, at room temperature for 4 d. The seeds were dehydrated through a graded ethanol series, gradually infiltrated with ethanolic LR White resin (London Resin Co., London, UK) into 100 % LR White, passed through three changes of 100 % LR White, stored at 4 °C for several weeks to complete infiltration and then embedded in LR White by polymerization under UV light in a nitrogen-flow cabinet. Thin sections (1·0 µm) were cut on a microtome from 4–5 seeds per species and mounted on glass slides prior to staining.
Histology
Semithin sections of LR White-embedded material were stained with 0·025 % Toluidine Blue in 0·05 M acetate buffer (pH 4·4), 1 % Amido Black 10B in 7 % acetic acid for proteins, Periodic Acid-Schiff (PAS) reaction (blocked for 30 min with 2,4-dinitrophenyl hydrazine in 15 % acetic acid) for carbohydrates with 1,2 glycol groups, and 0·023 % (w/v) aqueous (aq.) Calcofluor White M2R (CI 40622, Sigma, St Louis, USA) for ß-linked glucans including cellulose, following the methods of O'Brien and McCully (1981).
Handcut sections of unfixed material made in the transverse or medial longitudinal plane were stained with the following dyes: Toluidine Blue, pH 4·4, and Calcofluor White, Sudan Black B for total lipids (see above), 100 µg mL–1 Nile Red for neutral lipids (Oparka and Read, 1994), 0·02 % (aq.) Ruthenium Red for pectins and pectic acids, Chlor-Zinc-Iodide solution for cellulose/lignin/suberin, Acidic Phloroglucinol for lignin, and 1·0 % (w/v) Ferric Chloride in 0·1 M HCl for tannins, following the methods of Harris et al. (1994). Stained sections were mounted in water or glycerol. Freehand sections were also stained with 0·1 % (w/v, aq.) Berberine hemi-sulphate (Sigma, CI 75160) for 1 h then with 0·5 % (w/v, aq.) Aniline Blue for 30 min and mounted in glycerol–FeCl3 (0·1 %, w/v) for suberin (Barnabas, 1994), or were stained only with 0·1 % (aq.) Berberine solution for 1 h, rinsed in distilled water and mounted in water or glycerol. For confirmation of the presence of suberin, sections were covered with concentrated H2SO4 (Barnabas, 1996) for 27 h, rinsed, then stained with Sudan IV solution for 25 min, rinsed briefly with 70 % ethanol, mounted in glycerol and examined.
Movement of water to the embryo
While intact seeds take up water (Morris, 2000), it is not known whether water crossed the seed coat to the embryo. This was investigated by comparing the water content of embryos from imbibed and unimbibed seeds of one species only. Four seeds of G. sericea were placed into ten replicate Petri dishes lined with filter paper; five dishes were randomly selected and water was added. After 24 h, the embryos were dissected out, dried at 80 °C for 24 h and the water content calculated as a percentage of dry weight. Mean water content of imbibed and control seeds was compared by using a t-test.
Apoplasmic tracing
Six seeds from each species were immersed in 0·1 % (w/v, aq.) Lucifer Yellow CH (Molecular Probes L453 Lot 28C 1-5) for 24 h, rinsed three times in distilled water, blotted, sectioned with a safety razor blade and examined unmounted with an Olympus epifluorescent microscope using the 4x objective. Additional seeds were immersed in MilliQ water for the same amount of time to determine autofluorescence.
Fluorescence microscopy
Sections stained with Calcofluor White M2R and Berberine hemi-sulphate were examined under UV excitation (excitation filter UG.1, dichroic mirror 400 nm, barrier filter 420 nm). Sections stained with Nile Red and Lucifer Yellow CH were examined under blue excitation (excitation filter 490 nm, dichroic mirror 500 nm, barrier filter 515 nm). Unstained, unfixed sections were examined under UV and blue excitation for autofluorescence.
Fluorescence photomicrography was mainly carried out on an Olympus BHC epifluorescence microscope using Fuji Xtra 400 ASA colour film. Prints were scanned with a Canon Scanner N1240 U USB using ArcSoft PhotoStudio 2000. All images of the apoplasmic tracing with Lucifer Yellow were taken via a water-cooled digital camera with exposures of 145, 193 and 269 ms. Images were assembled into plates using Adobe Illustrator 11.0.0.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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When seeds of all three species gave similar results, results are reported for only one species. Where differences were apparent, results are reported accordingly.
Gross morphology and anatomy of the mature seed
The dry seed was elongated along the main axis, with an elaiosome at the former placental (i.e. chalazal) end and revolute margins folded under the seed (Fig. 1A). Imbibition resulted in a generalized swelling of the seed and a conformational change, as the margins of the seed coat unfolded from under the seed to a more lateral position (Fig. 1B). The embryo, which consisted of two cotyledons, a plumule and radicle, completely filled the embryo sac and there was no endosperm remaining (Fig. 1C). Between the embryo and the elaiosome was a structural discontinuity, the hypostase (Fig. 1D). A single vascular trace was found through the central region of the elaiosome, stopping just before the hypostase. Unfortunately, the vascular trace did not often survive sectioning and dropped out leaving an empty space (Fig. 1D). The radicle and plumule occupied a small amount of space at the micropylar end (Fig. 1E).
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Internal structure of the seed coat
Exotesta
The exotesta covering the lateral regions, upper surface, chalazal and micropylar regions of the seed consisted of small, squat, polygonal-shaped cells (Fig. 1C, D, F and G) but along the lower surface of the seed, between the two revolute lateral margins (wings in Fig. 1C), the cells were radially elongate, i.e. columnar in shape (Fig. 1H).
Mesotesta
Immediately beneath the squat exotestal cells there were 3–4 cell layers of thin-walled pigmented cells (Fig. 1C, D, F, G and I); the number of cell layers increased beneath the columnar exotestal cells (Fig. 1C) and at the micropylar end (Fig. 1E) where they abutted the wing-like extension of the endotesta.
Endotesta layer
The endotesta surrounded the lateral sides of the embryo sac (Fig. 1C) and continued into the elaiosome as far as the elaiosome apex (Fig. 1D). It consisted of thin-walled, palisade-shaped cells with a lace-like fibrous reticular network filling the interior (Fig. 1C, E, I and J). It was one cell thick around the embryo sac (Fig. 1C) and elaiosome [Figs 1D (inset a) and 2A], but in the ‘wing-like’ regions it was folded back on itself so that it appeared two cell layers thick (Figs 1C and 2B) and the radial and inner tangential cell walls were much thicker (Fig. 2B). The palisade cells of the endotesta did not extend across the elaiosome apex nor across the hypostase (Fig. 1D). In cross-section, small square-shaped areas occurred close to the outer tangential wall. These areas represent the location of crystals.
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Tegmen
The tegmen consisted of 2–3 cell layers of elongated, thick-walled cells (Figs 1I and J. and 2C–F). Although non-lignified (see below under Histology), we have still termed these cells ‘sclerenchyma’ following the broad definition of Esau (1960). In transverse sections through the middle of the seed, the long axis of the exotegmic sclerenchyma cells was parallel to the long the axis of the seed, while the second, endotegmic layer was perpendicular to the first, running around the seed. In the recurved margins of the seed, the tegmen was wedge-shaped (Fig. 2C, E and F). In longitudinal sections, the tegmen formed another wedge-shaped region around the micropylar end of the embryo sac (Fig. 1E) and at the chalazal end extended just past the end of the embryo sac to line the sides of the hypostase, ending there (Fig. 1D, inset b). The sclerenchyma cells were not continuous across the hypostase (Fig. 1D, inset b). A cuticle occurred on either side of the tegmen (Fig. 2D).
Hypostase
The hypostase consisted of brown-pigmented cells that could be grouped into two distinct regions. The innermost region abutting the embryo sac consisted of irregular, tessellated cells and the outer region abutting the elaiosome consisted of less distorted cells (Fig. 2G). The number of cells forming the hypostase varied between the three species; G. buxifolia had the greatest number of cell layers (i.e. 
20), with the other two species having 14–16 layers.
Elaiosome
The elaiosome consisted of a central region of large, variously shaped thin-walled cells bounded by an ‘epidermis’-like layer (Figs 1D, inset a, and 2A) that started at the hypostase and extended along the sides of the central region with short, bilayered extensions into the central region (Fig. 1D). As mentioned above, outside this central region were the endotesta, mesotesta and exotesta layers.
Crushed cell layer
A glutinous layer of crushed cells occurred between the endotegmen and the lateral sides of the embryo.
Histochemistry of the seed coat
Fresh and fixed/embedded samples were examined unstained and stained with several bright-field and fluorescent dyes to investigate the composition of the cell walls, vacuolar content and the presence/absence of cuticles. Although staining intensity and colour depths varied according to the thickness of the section, similar staining reactions were found in all three species for each of the different cell layers and types. All descriptions apply to all three species unless indicated otherwise.
Exotesta
The vacuoles of squat exotestal cells contained a brown substance that appeared sky blue in fixed (Fig. 1F) and fresh (not shown) material when stained with Toluidine Blue at pH 4·4 (indicating phenols), was positive with Amido Black, weakly positive with PAS and slightly coloured brown with Chlor-Zinc-Iodide (Fig. 1G), but was negative for tannins when stained with FeCl3 (Table 1). Irregular lines ran throughout the centre of the cells. These lines stained darker with Toluidine Blue (Fig. 1F) and Chlor-Zinc-Iodide (Fig. 1G). This material was not found in the columnar exotestal cells.
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The walls of the squat exotestal cells (in both fresh and fixed material) exhibited various staining reactions with Toluidine Blue at pH 4·4. The outer tangential and radial walls stained purple but the inner tangential walls stained blue (Fig. 1F). The walls stained purple with Chlor-Zinc-Iodide (Fig. 1G), stained positively with Amido Black, PAS, Ruthenium Red, Calcofluor White, Nile Red, Berberine hemi-sulphate and the Berberine/Aniline Blue combination (Tables 1
–3). They also stained with Sudan IV both before and after prolonged reaction with H2SO4, confirming the presence of suberin.
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With Toluidine Blue at pH 4·4, the outer tangential walls and outer part of the radial walls of the columnar exotestal cell stained purple, indicating the presence of polyanions, whereas the inner half (or base) of the radial walls were blue, indicating phenols (not shown). All walls of the columnar exotestal cells stained with Amido Black (weakly), Ruthenium Red (Fig. 1H) and PAS reagents (Table 1) and were autofluorescent white under UV illumination (Fig. 2H). In G. linearifolia and G. sericea, the entire wall stained with Calcofluor White, but in G. buxifolia only the tangential walls reacted (Table 2). After staining with Berberine hemi-sulphate, or the Berberine/Aniline Blue combination, the walls fluoresced an intense yellow (Fig. 2I), indicating polyanions and phenols. After Nile Red they stained emerald green (Fig. 2J) (Table 3) and after acid/Sudan IV they were red, indicating suberin. The cuticle over the surface of the exotesta reacted for lipid (orange) with Nile Red (Fig. 2J).
Mesotesta
The vacuoles of the mesophyll cells forming the mesotesta contained an ecru- to honey-coloured pigment (Fig. 2F) that was unstained by Chlor-Zinc-Iodide solution (Fig. 1G). Following staining with Toluidine Blue, the pigment was brown/blue (Fig. 1F and I), suggesting phenolic material. There was no reaction with FeCl3 (indicating an absence of tannins) nor with any other stain tested (Tables 1 and 3). The walls of the mesophyll cells in all species stained with Amido Black, PAS and Ruthenium Red. In G. linearifolia and G. sericea they stained purple with Toluidine Blue but gave a blue reaction in G. buxifolia (Table 1). The latter indicates incorporation of phenols into the walls, but this was not lignin because walls of all species did not react with Phloroglucinol–HCl. Following staining with Berberine hemi-sulphate solution, in G. buxifolia the walls in the outer cell layers of the mesotesta fluoresced gold (Fig. 2I) but the innermost layers abutting the endotesta were not fluorescent. In G. sericea the walls fluoresced a weaker yellow, and in G. linearifolia they were not fluorescent. With the Berberine/Aniline Blue combination there was no reaction from the walls of mesophyll cells beneath squat exotestal cells although there was some fluorescence from cells abutting the columnar exotestal cells in G. sericea, but not in G. linearifolia. Following staining with Nile Red, the walls were emerald green (Fig. 2J, Table 2).
Endotesta
Cells of the palisade-shaped endotesta stained somewhat differently depending on whether they were located close to the embryo, in the margins of the seed coat or around the elaiosome. The lace-like fibrillar network of the cells close to the embryo and in the wing region appeared yellowish outlined by blue–green when stained with Toluidine Blue (Fig. 1I). The contents of cells abutting the elaiosome stained blue with Toluidine Blue (Fig. 1D, insert a). The cell contents did not react with any other stains tested except for Amido Black, which outlined the lace-like fibrillar network (Table 1). The cell walls also gave varying staining reactions depending upon cell location. Where the endotesta was single layered, the walls appeared yellow after Toluidine Blue staining (Figs 1I and 2C), but in the wing-like regions, the radial and inner tangential walls of the cells stained turquoise with Toluidine Blue (not shown). Around the elaiosome, the radial walls were slightly thicker in G. buxifolia and stained blue–green (Fig 1D, inset a) but in G. linearifolia and G. sericea these walls were thinner and stained yellow (not shown). However, the radial walls of all endotestal cells reacted positively with Phloroglucinol–HCl, as did the inner tangential walls of cells in the wing-like region (Fig. 2B) and those abutting the elaiosome (Fig. 2A). After Chlor-Zinc-Iodide, the radial walls of all endotestal cells were yellow. Following Berberine/Aniline Blue staining the walls in all endotestal cells fluoresced yellow but when stained by Berberine hemi-sulphate alone, only the elaiosome and wing endotestal cells gave a positive reaction (greeny yellow) (Fig. 2K). The endotestal walls did not react with other stains (Table 1), except for Calcofluor White, which gave a slight increase in fluorescence (Fig. 2L) compared with autofluorescence in controls (Fig. 2M), and Nile Red, which gave green fluorescence in (Table 3).
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Tegmen
The thick cell walls of the tegmen stained deep blue with Toluidine Blue (Fig. 1C and I), pink with PAS (Fig. 1J) and Ruthenium Red, and blue–black with Amido Black but were negative with all other bright-field stains especially those testing for lignin (Table 1). They were only partially positive for ß-glucan (probably cellulose), except in the wedge-shaped region where a stronger reaction was found in the radial walls (Table 3). Following staining with Berberine hemi-sulphate alone, the walls fluoresced golden amber but stained negatively after the Berberine/Aniline Blue combination.
There was a thick cuticle between the exotegmen and the endotesta, particularly around the wedge-shaped areas (Fig. 2D and E), and between the endotegmen and the crushed cell layer there was a thin and sometimes discontinuous cuticle. The cuticles were autofluorescent and gave a positive reaction with Nile Red (Fig. 2D and E) and with Sudan Black B (not shown).
Crushed cell layer
The amorphous material lying between the endotegmen and the embryo stained blue with Toluidine Blue (Fig. 1I) but only gave a positive reaction with PAS (Fig. 1J), Amido Black (Fig. 2F), Ruthenium Red (Table 1) and Calcofluor White (Table 2, 3).
Hypostase
In the hypostase cells, the brown pigment in the vacuoles suggested the presence of tannins, but there was no positive reaction with FeCl3 in any seed. Only the cell walls of the inner 5–7 layers of cells immediately adjacent to the cotyledons stained positively with Sudan Black for total lipids (Fig. 2G). Nile Red-induced fluorescence for neutral lipids (Fig. 2N, Table 4) varied but was impossible to interpret because the walls of the inner layers of the hypostase were strongly autofluorescent (canary yellow) under blue excitation. When seeds were pre-stained with Phloroglucinol–HCl and examined under UV and blue excitation, only the radial walls of the endotestal cells lost their autofluorescence. When counterstained with Chlor-Zinc-Iodide solution, there was a diminution of UV autofluorescence in the cell walls of the inner layers of the hypostase, indicating that suberin may be present (not shown). The greatest loss of autofluorescence occurred in G. sericea, followed by G. linearifolia. There was only minimal loss of autofluorescence in G. buxifolia. Following staining with Berberine hemi-sulphate the walls of the innermost cell layers fluoresced brightly yellow (Table 4) but the outer five layers (i.e. next to the elaiosome) did not (not shown).
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Histochemistry of the central elaiosome cells and embryo
Central elaiosome cells
The thin walls of cells of the central region of the elaiosome stained purple with Toluidine Blue (Fig. 1D), and gave a positive reaction with PAS, Calcofluor White and Berberine hemi-sulphate (Tables 1, 4); the cell contents stained with Sudan Black, Nile Red (lipids) and Berberine hemi-sulphate (not shown), and the cytoplasm was positive with Amido Black (not shown).
Embryo
The cells of the cotyledons contained numerous storage bodies that were negative for starch when stained by the PAS reaction (Fig. 1J) but were positive for proteins (Fig. 2G) and lipids (not shown).
Movement of water to the embryo
The water content of embryos from seeds of G. sericea that had imbibed for 24 h (mean 30·4 ± 0·5 %) was about six times greater than that of embryos from dry seeds [mean 5·3 ± 0·02 %; comparison of (log-transformed) means, t8d.f. = 100·6, P < 0·001].
Apoplasmic tracing with Lucifer Yellow CH
Seeds examined after soaking for 24 h in Lucifer Yellow (LY) showed a variable fluorescence that differed from autofluorescence in the controls. In all species, there was strong lemon-yellow fluorescence indicative of LY in the outer tangential and radial walls of the columnar and squat exotestal cells in all regions of the seed (Fig. 3B–D, F–I, K, M, O, Q, S, T and V). This was quite distinct from the dull more olive-green autofluorescence of these tissues in the controls (Fig. 3A, E, J, L, N, P, R and U). In two G. buxifolia seeds (Fig. 3H and I) and one G. sericea seed, there was also LY-induced fluorescence in the adjacent mesophyll cell layers and in one G. buxifolia seed section the radicle (Fig. 3G) and cotyledons (Fig. 3I) also exhibited LY-fluorescence. In other replicates of these species there was no LY-fluorescence in the embryo or mesophyll cell walls. Lucifer Yellow consistently accumulated around the oil-filled thin-walled elaiosome cells in G. sericea (compare Fig. 3N and O), and in one replicate of G. linearifolia (not shown). Lucifer Yellow also accumulated in certain cells in the micropylar region (compare Fig. 3P and Q); the origin of these cells is unknown. No LY-fluorescence was found in any of the cells of the endotesta or tegmen (Fig. 3G, K, O, S, T and V).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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Seed coat structure
The anatomy of the seed coat of the three Grevillea species examined in this study is consistent with previous descriptions, particularly those by Corner (1976)
, for Grevillea species and the Proteaceae. The presence of the two internal cuticles, one on either side of the sclerenchyma layer, confirms that the bi-layered sclerenchyma is the tegmen and the inner palisade layer with the fibrillar contents and crystals represents the endotesta. The layer of crushed cells between the tegmen and the embryo may be the remains of the nucellus (Venkata Rao, 1967; Corner, 1976), although a study of the development of these seeds is needed to confirm this. The revolute margins of the seeds appear to be formed by the folding of the testa and tegmen, as evidenced by the expansion and folding of the single-layered palisade-like endotesta into the double-layered palisade forming the ‘wing’; the formation of the sclerenchyma ‘wedge’ indicates that the tegmen has been similarly folded. The highly tessellated cells lying at the chalazal or elaiosome end of the seed clearly represent a hypostase, as the sclerenchymatous tegmen extends along the lateral edges, but not across this zone and the zone lies between the elaiosome and the embryo sac. The columnar exotestal cells and adjacent mesophyll cells constitute the ‘narrow wing’ of the oat-style seed. Our study of the anatomy and histochemistry of the seed coat has identified a number of potential dormancy mechanisms. These will now be considered in turn.
Barriers to diffusion
We observed an exterior cuticle, and two internal cuticles on either side of the tegmen. Cuticles over the surfaces of leaves, fruits and seeds consist of a cutin membrane overlain with waxes. The semi-permeable nature of cuticles is thought to permit the inward diffusion of water and oxygen (Bewley and Black, 1982), but prevents the outward flow of endogenous inhibitors such as polyphenolics (Swain, 1979, cited in Egerton-Warbuton, 1998). A comparison of the water content of embryos from imbibed and dry seeds of G. sericea indicated that water crosses the seed coat and reaches the embryo; thus, the internal cuticles are permeable to water.
The cuticles identified in the seed coat of Grevillea are potential sites for action by smoke (Egerton-Warburton, 1998). In the seeds of the chaparral species Emmenanthe penduliflora, a post-fire recruiter, Lucifer Yellow CH (LY) penetrated the external cuticle but was stopped by the internal cuticle that separated the seed coat from the endosperm. The inner cuticle of dormant seeds of E. penduliflora had pores that allowed movement of water into and out of the endosperm, but not of larger molecules such as Lucifer Yellow CH (molecular mass 457 Da). However, after treatment with smoke, the permeability of the inner cuticle increased and LY penetrated into the endosperm and embryo, apparently due to an increase in the number and diameter of the cuticular pores (Egerton-Warbuton, 1998). A sucrose ester that inhibited germination was subsequently identified from seeds of E. penduliflora; smoke thus broke dormancy by allowing the inhibitor to escape the seed (Egerton-Warburon and Ghisalberti, 2001).
Our study with LY on untreated Grevillea seeds identified several barriers to diffusion of high-molecular-weight compounds. In the three species, although the external cuticle was no barrier to the movement of LY, there was a major barrier located at the base of the exotesta (i.e. the basal portion of the radial walls and the inner tangential walls) that effectively prevented the inward flow of large molecules. The barrier effect of the exotestal cells to LY may have been due to suberization and/or incorporation of phenols into the walls. The presence of suberin in the exotestal cell walls of these three Grevillea species was demonstrated following staining with Berberine hemi-sulphate and confirmed by acid hydrolysis followed by staining with Sudan IV. Berberine hemi-sulphate is an acid salt of the alkaloid berberine, which has been used supposedly to confirm the presence of suberin in plant roots (Barnabas, 1996). However, as a cationic dye it will also bind to polyanions and so will bind to phenolic compounds.
Another barrier to diffusion of high-molecular-weight compounds was identified at the endotesta because although LY was able to penetrate through the central elaiosome region in G. sericea it did not spread laterally into the abutting endotesta. The endotesta thus acted as a second barrier to the inward/outward movement of large molecules such as germination inhibitors. This situation was paralleled at the micropylar end, where LY penetrated the short distance through the thin-walled micropylar cells but was stopped at the endotesta. Hence, if the exotestal layer becomes disrupted due to wear and tear, the endotesta may still impose dormancy by regulating movement of germination inhibitors present in embryo or seed coat. The palisade layer in Glycine seeds has been identified as the site of the barrier to diffusion (of water; Ma et al., 2004).
The endotesta layer is not continuous across the hypostase; however, Lucifer Yellow did not cross the hypostase to the embryo, indicating a barrier to diffusion in the hypostase. The suberin identified in the cell walls of the innermost cell layers of the hypostase may act as a barrier to movement of inhibitors to and from the embryo.
The embryo would thus appear to be completely surrounded by an outer and an inner barrier to the diffusion of high-molecular-weight compounds. Whether these barriers play a role in dormancy, for example by restricting the exit of germination inhibitors from the embryo, and whether smoke and/or heat shock modify this as in E. penduliflora still remain to be tested.
Phenolic compounds—inhibitors of gas exchange
Phenolic compounds have been proposed to inhibit seed germination (Bewley and Black, 1982; Egerton-Warburton, 1998). The presence of phenolic compounds in the radial walls of the columnar exotestal cells and in the inner tangential walls and vacuoles of the mesotestal cells was indicated by the autofluorescence and blue reaction with Toluidine Blue. Phenolic compounds such as coumarin, chlorogenic acid, ferulic acid, and caffeic and sinapic acid (and their derivatives) are esterified to wall carbohydrates (Pan et al., 2002) and can be found in the seed coat and other seed tissues in many species (Gubler and Ashford, 1985). The strongly negative environment in all the walls of the squat exotestal cells and the outer half of the columnar exotestal walls is indicated by the metachromatic reaction of Toluidine Blue at pH 4·4, suggesting polyanions (O'Brien and McCully, 1981). (They are also likely to be present in other regions but were masked by the blue reaction for phenols.) These conclusions are supported by the positive reaction for polyanions with Ruthenium Red (Webster and Stone, 1994) and demonstration of acid residues of the phenolic compounds by Berberine hemi-sulphate. This would be expected to result in a very hydrated system following wetting, and it is upon wetting of the seed coat that phenolic compounds undergo the oxidation that is reported to interfere with gas exchange between the embryo and the environment (Bewley and Black, 1994). The phenolic compounds identified in the exotestal cell walls could thus potentially interfere with gas exchange.
The normal staining response of Toluidine Blue, pH 4·4, given by lignified walls was only found in cells forming the expanded wing area of the endotesta, not elsewhere in the endotesta in these seeds. (Toluidine Blue at pH 4·4 produces a turquoise colour when it binds to lignin in the secondary walls of xylem vessels). A turquoise colour only occurred in some of the walls; most walls were yellow as in controls of embedded material. However, when acidified phloroglucinol was used, the entire endotestal layer gave a positive response for lignin, albeit only in the radial and inner tangential walls; no other cell wall gave a positive result with this dye. Staining variability may be due to masking of lignin by other phenolic compounds, but is most likely to be caused by variability in access of the dyes to staining sites, a problem encountered particularly with fresh material.
Mechanical restraint
Seeds of the Proteaceae are endotestal, i.e. the region of chief mechanical restraint is the lignified (or unlignified) palisade cells of the endotesta and the adjacent fibrous tegmen, as was found in the three Grevillea species investigated here. Another potential region of mechanical restrain could be the exotestal cells due to deposition of phenols and suberin in the cell walls.
The discontinuity of the exotesta and tegmen across the hypostase could be important if the seed coat is acting as a mechanical restraint: it gives a point of structural difference in the seed coat, which could potentially be affected by germination cues such as heat shock.
Unrolling of margins during hydration
The structural reinforcement of the sclerenchymatous tegmen and lignified endotesta also appears to be responsible for the unrolling of the recurved margins during imbibition. When G. sericea sections were compared before and after hydration, the region between the recurved margins doubled in width in the direction between the palisade and the columnar epidermal cells and increased by 72 % in the direction between the margins themselves. No change in the width of the seed coat was found in the rest of the seed. Expansion of the seed in the columnar exotestal and mesotestal cells would force the rolled margins outward. The deposition of phenolic compounds in the radial walls of the columnar exotestal and the endotestal cells would result in a tangential expansion of these cells whilst the absence of phenolic compounds in the walls of the mesophyll cells would permit both tangential and radial expansion.
Nile Red staining of cell walls: a novel histochemical result
Nile Red was used to confirm the presence of neutral lipids such as formed in the cuticles mentioned above. However, an unexpected emission colour, emerald green (500–550 nm), was observed when the walls of the exotestal, mesotestal and elaiosome endotestal cells stained by Nile Red were excited by blue light. The normal emission range of Nile Red is c. 100 nm, i.e. from red (660–680 nm) to yellow (550–580 nm). The emerald green emission colour represents an extra shift of at least 30 nm, towards the blue end of the spectrum. Nile Red is an uncharged heterocyclic planar hydrophobic phenoxazine dye (Fig. 4) (Sackett and Wolf, 1987; Haugland, 2002). It exhibits solvatochromism, i.e. its emission maximum varies depending on the hydrophobicity of its environment. In polar solvents it appears red but undergoes a blue shift as the polarity of the solvent decreases. Because of its hydrophobic properties, Nile Red partitions not only into neutral lipids but also into hydrophobic regions of lipoproteins (Greenspan and Fowler, 1985), native proteins (Sackett and Wolf, 1987) and alpha-acid glycoprotein (Brown et al., 1995). The observed blue shift seen in the walls of the exotestal and endotestal cells may be due to high hydrophobicity of the wall; it may also be due to an acid–base interaction between the dye's ketone side group and wall acids. If the ketone side group of the dye is protonated, there will be a shift in the conjugation of the double bonds. When the conjugation of double bonds is altered in cyclic molecules there is spectral shift in absorbance and emission; for example, when cyclic ß-diketones are placed into alkaline solutions there is a change to the enolate form with the formation of a single double bond in the ring. The formation of this double bond results in a 30-nm red shift in absorbance (UV illumination) (Silverstein et al., 1991). Therefore, when the double bond is removed or shifted from a conjugated system there should be a blue shift of 30 nm. To determine whether a blue shift occurs in Nile Red following acidification, a simple test was carried out with Nile Red and 10 % acetic acid, and between Nile Red and a crystal of pure phenol. Both the acid and the phenol caused strong blue shifts (purple with acetic acid and deep blue with phenol) in the colour of the dye when viewed under white light. These shifts were large enough to explain the shift from orange to green observed in the Grevillea walls and thus to support the acid–base hypothesis.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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In summary, Grevillea linearifolia, G. buxifolia and G. sericea show almost identical staining reactions with the dyes used. The slight differences may contribute to the observed differences in dormancy of collected seed, and to differences in germination response to heat/smoke treatments (Kenny, 2000
; Morris, 2000). Differences between the three species were most marked in the hypostase, which may determine the dormancy and germination characteristics of each species. These three Grevillea species possess potentially three of the five criteria outlined by Bewley and Black (1982) for seed-coat imposed dormancy: mechanical restraint, possible interference with gas exchange and possible prevention of the exit of inhibitors from the embryo. One criterion that could not be determined was the supply of inhibitors to the embryo. The influence of the internal cuticles on movement of large molecules, as shown for Emmenanthe penduliflora (Egerton-Warbuton, 1998), could not be determined because of the barriers imposed on the diffusion of Lucifer Yellow by the epidermis and the palisade. |
ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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We thank Dr Mark Williams, University of Western Sydney, Hawkesbury, for his assistance in explaining the colour shift shown by Nile Red when applied to the exotestal and endotestal cell walls.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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