The Emergence of Embryos from Hard Seeds is Related to the Structure of the Cell Walls of the Micropylar Endosperm, and not to Endo-β-mannanase Activity

Abstract

• Background and Aims Seeds of carob, Chinese senna, date and fenugreek are hard due to thickened endosperm cell walls containing mannan polymers. How the radicle is able penetrate these thickened walls to complete seed germination is not clearly understood. The objective of this study was to determine if radicle emergence is related to the production of endo-β-mannanase to weaken the mannan-rich cell walls of the surrounding endosperm region, and/or if the endosperm structure itself is such that it is weaker in the region through which the radicle must penetrate.

• Methods Activity of endo-β-mannanase in the endosperm and embryo was measured using a gel assay during and following germination, and the structure of the endosperm in juxtaposition to the radicle, and surrounding the cotyledons was determined using fixation, sectioning and light microscopy.

• Key Results The activity of endo-β-mannanase, the major enzyme responsible for galactomannan cell wall weakening increased in activity only after emergence of the radicle from the seed. Thickened cell walls were present in the lateral endosperm in the hard-seeded species studied, but there was little to no thickening in the micropylar endosperm except in date seeds. In this species, a ring of thin cells was visible in the micropylar endosperm and surrounding an operculum which was pushed open by the expanding radicle to complete germination.

• Conclusions The micropylar endosperm presents a lower physical constraint to the completion of germination than the lateral endosperm, and hence its structure is predisposed to permit radicle protrusion.

INTRODUCTION

Following pollination in angiosperms, fertilization of the egg cell results in the zygote, which, though a series of mitoses, gives rise to the embryo. In addition, and unique to the angiosperms, a second fertilization of the polar nuclei in the central cell produces the endosperm mother cell. Mitotic divisions of this, typically triploid, cell produces the endosperm. At maturity, the seeds of angiosperms are variable in structure, but generally consist of the embryo surrounded and protected by the testa. The fate of the endosperm, however, varies depending on species. It may persist as a major storage structure (e.g. cereals, Ricinus communis, endospermic legumes) or as a thin layer, one to a few cells thick, surrounding the embryo (e.g. Lactuca sativa, Gossypium herbaceum, Glycine max, Arabidopsis sp.) or it may be completely absorbed during seed development and hence be absent from the dry seed (e.g. Pisum sativum, Phaseolus sp., Aesculus hippocastanum; Bewley and Black, 1978; W. Stuppy, Royal Botanic Gardens, Kew, pers. comm.).

In mature seeds such as lettuce, tomato and Datura, the cells of the endosperm have thickened cell walls rich in mannan-based polymers. These polymers serve as a carbohydrate store to support post-germinative seedling growth. After imbibition of the seeds, however, the endosperm also obstructs germination by acting as a physical barrier to radicle protrusion. Hence, it is generally accepted that weakening of the endosperm, particularly in the micropylar region adjacent to the radicle, is a prerequisite for the completion of germination. This weakening is initiated by the hemicellulase, endo-β-mannananse (Bewley, 1997), although activity of this enzyme alone may not be sufficient to ensure germination in all species (Bewley and Halmer, 1980/81; Nonogaki et al., 1992; Sànchez and de Miguel, 1997; Bradford et al., 2000).

Seeds of many species are extremely hard in the mature dry state because the walls of the endosperm cells are heavily thickened with galactomannan or mannan polymers. In date and coffee, the extreme hardness is conferred by the presence of water-insoluble crystalline mannans in the endosperm cell walls (Bewley and Reid, 1985). The endosperm walls in carob (McCleary and Matheson, 1974) and fenugreek (Reid, 1971) contain hydrophilic galactomannans which become mucilaginous after a period of imbibition and usually some hydrolysis (Reid and Bewley, 1979). Chinese senna seeds also become mucilaginous with time of imbibition.

The question arises as to how germination, terminating with the protrusion of the radicle, is able to proceed in seeds where the embryo is enveloped by an extremely hard endosperm. Two possibilities exist: (1) the walls of the endosperm cells in the micropylar region are weakened by hydrolytic enzymes such as endo-β-mannanase prior to the completion of germination or (2) the structure of the endosperm in the micropylar region is modified and is less resistant to radicle penetration, most likely through a modification of cell wall structure and/or composition. Previous reports on the germination of two very hard seeds, Asparagus officinalis and Coffea arabica (Williams et al., 2001; da Silva et al., 2005) have established that morphological differences exist between the cells of the micropylar and lateral endosperm, the former region of the endosperm possessing considerably thinner walls than the latter. Thus the anatomical structure of the endosperm in extremely hard seeds can be such that germination is allowed to proceed without constraint. This led to this study to determine if such structural differences also exist in other seeds that have a hard endosperm at maturity. Seeds of fenugreek, carob, Chinese senna and date were chosen as the main species for study, to test the hypothesis that the structure of the micropylar endosperm, with respect it having thin cell walls, is more important in germination than the activity of the hemicellulase, endo-β-mannanase to disrupt the integrity of the constitutive mannans.

MATERIALS AND METHODS

Plant material

Fenugreek (Trigonella foenum-graecum L.) and caraway (Carum carvi L.) seeds were purchased from the Stone Store (Guelph, ON, Canada). Seeds of Chinese senna (Cassia tora L.) and carob (Ceratonia siliqua L.) were purchased from Richters Co. (Greenwood, ON, Canada). Date (Phoenix dactylifera L.) seeds were purchased from Caterseeds Co. (Goleta, CA, USA). Coffee (Coffea arabica L.) seeds were a gift from Dr Amaral da Śilva, UFLA, Lavras, Brazil.

Seeds of fenugreek and Chinese senna were germinated on Whatman No. 1 filter paper in 9-cm-diameter Petri dishes (Fisher Scientific Ltd, Nepean, ON, Canada) on 2 mL deionized water at 25 °C in the dark. Seeds of carob and date were germinated on sterile cotton in 9-cm-diameter Petri dishes on 10 mL deionized water at 25 °C in the dark. Owing to their extreme impermeability, the seed coats of carob and Chinese senna were nicked at the cotyledon end before sowing to allow for the imbibition of water. For the germination time course, duplicates of 25 seeds of fenugreek and Chinese senna were placed on 5 mL deionized water, for date duplicates of five seeds on 15 mL water, and for carob ten seeds on 15 mL water.

Endo-β-mannanase (EC 3.2.1.78) extraction and assay

Duplicate lots of five dissected seed parts (embryo, micropylar endosperm and lateral endosperm) of carob and date, and ten of Chinese senna and fenugreek were ground in an ice-cold mortar and pestle with different volumes of 0·1 m HEPES–KOH buffer, pH 8 (Sigma Aldrich Canada Ltd, Oakville, ON, Canada) and a small amount of washed sea sand to extract endo-β-mannanase (Toorop et al., 1996). After centrifugation at 21 000 g at 4 °C for 5 min, the supernatants were assayed in duplicate for endo-β-mannanase activity using a modified gel diffusion assay (Bewley et al., 2000). Activity was expressed in relation to the activities of a serial dilution of Aspergillus niger endo-β-mannanase (Megazyme, Bray, Eire) as a standard (Downie et al., 1994).

Light microscopy

Whole seeds of fenugreek, Chinese senna and caraway (imbibed 6 h), half seeds of carob (imbibed 2 d), and 2- to 3-mm-thick longitudinal slices of date and coffee seeds (imbibed 2 d) were fixed in 2 % (v/v) acrolein and 3 % (v/v) glutaraldehyde in 25 mm Na phosphate buffer (pH 7·2) at 4 °C for 4 d with gentle shaking (Brown and Greenwood, 1990; Williams et al., 2001). Seeds or seed parts were washed with distilled water and then dehydrated through a graded ethanol series: 30 % and 50 % (v/v) ethanol/water for 12 h each, 70 %, 80 % all at 4 °C, then 90 % and 95 % ethanol/water for 24 h each, and then through three changes for 100 % ethanol over 3 d all at room temperature.

Following dehydration, seeds or slices were incubated in 50 : 50 fresh 100 % ethanol : propylene oxide (BDH, Toronto, ON, Canada) at room temperature for 1 d, followed by pure propylene oxide for 1 d. Samples were then placed in 3 : 1 propylene oxide: Spurr's epoxy resin (hard mixture; Spurr, 1969) for 1 d. This was followed by 50 : 50, then 25 : 75 propylene oxide : Spurr's resin for 2 d each, then pure resin was exchanged each day for 4 d prior to polymerization at 65 °C overnight.

Sections, 2–3 µm thick, were cut with a Sorvall Porter-Blum JB4 microtome with wet glass knives, and then mounted on Colourfast microscope slides stained with 0·5 % (w/v) toluidine blue in 0·1 % (w/v) sodium borate, pH 8 (Williams et al., 2001). All observations were made using a Zeiss Jena Jenalumar contrast compound microscope under bright-field and polarized light, and images taken with a Nikon CoolPix 950 digital camera.

RESULTS

Fenugreek

Fenugreek seeds completed germination 24 h from the start of imbibition; negligible or no endo-β-mannanase activity was detectable in any of the seed parts (embryo, micropylar endosperm and lateral endosperm) over this period (Fig. 1). By 36 h, enzyme activity was observed in the micropylar and lateral endosperm but was barely detectable in the embryo. Enzyme activity was higher in the micropylar endosperm than in the lateral, but decreased substantially by 72 h in the former due to its general decomposition following penetration by the radicle.

Fig. 1.

Endo-β-mannanase activity in the different tissues of fenugreek seeds during and following germination (shown as a percentage). Standard error bars indicate the variability between duplicate assays of duplicate extracts.

The fenugreek seed is described as having a bent foliate embryo (Radford et al., 1974), the cotyledons lying adjacent to the radicle. At maturity, the relatively large embryo is completely surrounded by the endosperm (Fig. 2A). This in turn is surrounded by the seed coat having macrosclereids comprising the outer palisade layer (Fig. 2B). The testa in fenugreek does not appear to be resistant to penetration by the radicle at the completion of germination.

Fig. 2.

Structure of the fenugreek seed. (A) Transversely dissected seed showing the radicle, cotyledon, micropylar endosperm and lateral endosperm. (B) Lateral endosperm cells with thick cell walls. The outmost layer of the endosperm is the aleurone layer which contains the only living cells of the endosperm. (C) Micropylar endosperm region, where the endosperm is only a single layer of cells, the aleurone layer, adjacent to the radicle. A, aleurone layer; Co, cotyledon; LEn, lateral endosperm; MEn, micropylar endosperm; R, radicle; Sc, seed coat. Scale bars: A = 1 mm; B, C = 200 μm.

In fenugreek seed the outermost cell layer of endosperm, the aleurone layer, is complete and composed of relatively small, thin-walled, living cells (Fig. 2B and C). The lateral endosperm, lying adjacent to the cotyledons (Fig. 2B) and to most of the hypocotyl, comprises the majority of the endosperm tissue. Other than those of the aleurone layer, all cells in the lateral endosperm have extremely thick galactomannan- containing (Reid, 1971) walls (Fig. 2B), which occlude most of the cytoplasm. However, the micropylar endosperm cells surrounding the radicle tip have a distinctly different morphology from those of the lateral endosperm. The micropylar endosperm is comprised solely of the thin-walled cells of the aleurone layer (Fig. 2C).

Carob

Endo-β-mannanase activity in carob seed was low but measurable in all seed parts after 3 d of imbibition, but there was no change in activity until after the completion of germination (Fig. 3). Enzyme activity was higher in the micropylar and lateral endosperm over the 2 d following radicle emergence, but remained very low in the embryo even after the completion of germination.

Fig. 3.

Endo-β-mannanase activity in the different tissues of carob seeds during and following germination (shown as a percentage). Standard error bars indicate the variability between duplicate assays of duplicate extracts. G, germinated; NG, non-germinated.

At maturity, the embryo of carob has large flattened cotyledons and is completely surrounded by the endosperm, which is thicker on the flat plane of the cotyledons than at the cotyledon edges (Fig. 4A). Surrounding the endosperm, the testa is well-sclerified with an ordered outer palisade layer (Fig. 4B and C). It is also relatively impermeable to water and thus restricts germination.

Fig. 4.

Structure of the carob seed. (A) Longitudinal sagittal (left) and coronal (right) sectioned seeds showing the radicle, cotyledon and endosperm regions. (B) Lateral endosperm cells with thick cell walls and a thick seed coat to the outside. (C) The transition region between the micropylar endosperm cells with thinner walls and the lateral endosperm cells with thicker walls. The seed coat to the outside of the transition region and the radicle to the inside of this region. (D) The micropylar endosperm with thin-walled cells around the radicle, and the seed coat around the micropylar endosperm. Co, cotyledon; LEn, lateral endosperm; MEn, micropylar endosperm; R, radicle; Sc, seed coat; Tr, transition region. Scale bars: A = 2 mm; B–D = 200 μm.

The endosperm cells in carob remain alive following maturation drying of the seed, and there is no aleurone layer present. The walls of the lateral endosperm cells are thick due to the presence of galactomannans (Reid, 1985), and there is less cytoplasm in those cells distal from the radicle because of the thicker cell walls (Fig. 4B). There is an obvious transition between the lateral and micropylar endosperm with a decrease in cell size, wall thickness and number of cell layers in the latter (Fig. 4C). The micropylar endosperm is reduced to only four or five layers of thin-walled cells surrounding the radicle tip (Fig. 4D).

Chinese senna

There was very little detectable endo-β-mannanase activity in extracts from the different Chinese senna seed parts prior to the completion of germination; activity was higher in the micropylar and lateral endosperm extracts by the time 70 % of the seeds had completed germination at 36 h (Fig. 5). By 48 h, when all seeds had completed germination, endo-β-mannanase activity in the endosperm had markedly increased; activity in the embryo remained low even after germination.

Fig. 5.

Endo-β-mannanase activity in the different tissues of Chinese senna seeds during and following germination (shown as a percentage). Standard error bars indicate the variability between duplicate assays of duplicate extracts. G, germinated; NG, non-germinated.

At maturity, the embryo of Chinese senna has a relatively large radicle/hypocotyl and flattened cotyledons folded within the surrounding endosperm (Fig. 6A), which in turn is surrounded by a hard sclerified testa having an organized palisade layer (Fig. 6B and D). The testa is impermeable and restricts water uptake into the seed. Cotyledons and radicle/hypocotyl cells contain numerous protein storage vacuoles (Fig. 6B and C).

Fig. 6.

Structure of the Chinese senna seed. (A) Transversely dissected seed showing the radicle, cotyledon, micropylar and lateral endosperm. (B) The lateral endosperm cells with thick walls and the seed coat to the outside. (C) The transition region between the micropylar endosperm cells with thin cell walls and the lateral endosperm cells with thick cell walls. A cotyledon tip and the radicle are visible in this region. (D) The micropylar endosperm with thin-walled cells around the radicle. Co, cotyledon; LEn, lateral endosperm; MEn, micropylar endosperm; R, radicle; Sc, seed coat; Tr, transition region. Scale bars: A = 2 mm; B, D = 200 μm; C = 100 μm.

The cells in the endosperm are living and contain storage materials; no aleurone layer is present (Fig. 6B). With the exception of those lying adjacent to the testa, cells of the lateral endosperm have very thick cell walls (Fig. 6B). There is a transition region between the lateral and micropylar endosperm with the cells of the micropylar endosperm being smaller and having thin walls (Fig. 6C and D). Immediately surrounding the radicle tip, the micropylar endosperm comprises three layers of thin-walled cells (Fig. 6D).

Date

At 9 d after the start of imbibition, but prior to the completion of germination, a low amount of endo-β-mannanase activity was detectable in the embryo but not in the endosperm of date seed (Fig. 7). After the completion of germination (13 d), enzyme activity had increased in both the embryo and micropylar endosperm, but was barely detectable in the the lateral endosperm. Continued marked increases in endo-β-mannanase activity in the embryo and micropylar endosperm were noted for a further 4 d, but there was little increase in activity in the lateral endosperm (Fig. 7).

Fig. 7.

Endo-β-mannanase activity in the different tissues of date seeds during and following germination (shown as a percentage). Standard error bars indicate the variability between duplicate assays of duplicate extracts. G, germinated.

Unlike in the other seeds studied, the radicle region of the embryo is positioned in the centre of the date seed and lies beneath a small concave operculum on the testa that is marked by a circular furrow (Fig. 8A, upper). Cells of the testa are thin-walled (Fig. 8C) and the testa does not restrict water uptake. Completion of germination is marked by the radicle emerging through the testa and underlying endosperm with the operculum splitting away from the remaining testa along the furrow (Fig. 8A, lower).

Fig. 8.

Structure of the date seed. (A) Dry seed (upper) and germinated seed (lower). The circles indicate the operculum region of the seed coat in the micropylar area with the flap (arrowed) formed from a part of the micropylar endosperm pushed out by the protruding radicle following germination. (B) The lateral endosperm cells with thick cell walls which surround the embryo. (C) The micropylar endosperm with radicle. The arrows show the cells with thin walls which form a circle in the operculum region in micropylar area. Several layers of cells around the embryo are thin-walled and depleted. (D) Closer view of the micropylar endosperm around the embryo showing the region of cells with thin walls. Em, embryo; LEn, lateral endosperm; MEn, micropylar endosperm; R, radicle; Sc, seed coat. Scale bars: A = 2 mm; B–D = 200 μm.

The endosperm of the date seed is extremely hard, due to the endosperm cell walls being composed of galactomannan but having <10 % galactose side chains (Bewley and Reid, 1985). The endosperm cells are still living and contain storage materials (Fig. 8B). Unlike in fenugreek, Chinese cabbage and senna, thick walls are evident in both the lateral and the micropylar endosperm cells (Fig. 8B–D). However, while most of the cells in the micropylar endosperm region are thick-walled, there is a delimiting region overlying the radicle tip and extending through the micropylar endosperm to the endosperm periphery in which the cells are thin-walled (Fig. 8C and D). These thin-walled cells form a circle immediately underlying the furrow on the testa that defines the operculum (Fig. 8C and D).

Other seeds

Similar morphological differences in structure between the lateral and micropylar endosperm regions were also observed in caraway and coffee seeds (Fig. 9). In both species, the endosperm cells are living (Fig. 9). The lateral endosperm cells have thick walls in caraway (Fig. 9B), but the walls of the micropylar endosperm cells surrounding the radicle tip are much thinner (Fig. 9A), although the micropylar endosperm comprises a greater number of cell layers than in most of other seeds studied. Similarly, the micropylar endosperm cells of coffee seed have much thinner walls than those of the lateral endosperm (Fig. 9C).

Fig. 9.

Structure of caraway and coffee seeds. (A) The lateral endosperm of caraway seed, with thick-walled cells, and seed coat. (B) The micropylar endosperm with thin-walled cells around the radicle of the caraway seed with the seed coat to the outside. (C) The endosperm and a part of the radicle close to the tip of the coffee seed. The micropylar endosperm around the radicle has thin-walled cells and the lateral endosperm away from the radicle has thicker-walled cells. Even thicker-walled cells lie at the distal end of the endosperm (not shown). LEn, lateral endosperm; MEn, micropylar endosperm; R, radicle; Sc, seed coat. Scale bars = 200 μm.

DISCUSSION

The hard-seeded species used in the current study all possess a substantial endosperm with thickened cell walls composed of mannans and/or galactomannans. Because it completely surrounds the embryo, it is possible that the endosperm restricts or prevents germination owing to the nature of these walls. Indeed, it has been suggested that the weakening of the endosperm cell walls is required to allow for protrusion of the radicle from the seed (Bewley, 1997). The major enzyme responsible for the degradation of mannan-containing polymers present in cell walls is endo-β-mannanase. It is present in many seeds; in some it is active during germination, but more usually the activity of the enzyme is not detectable until after germination is complete (Dirk et al., 1995). In the species studied here, the enzyme is either absent or had minimal activity in either the embryo or the endosperm prior to the completion of germination. Thus the emergence of the radicle from the seed in these species is unlikely to be dependent upon the weakening of the major, mannan-rich components of the cell walls of the surrounding endosperm. There is the possibility that other hemicellulases which degrade the minor components of the walls play a role in germination, although this would seem to be less likely.

In both asparagus and coffee seeds, although the vast majority of cells in the lateral endosperm have thickened cell walls, those of the micropylar endosperm adjacent to and overlying the radicle tip have relatively thin walls (Williams et al., 2001; da Silva et al., 2005). This localized modification of endosperm cellular structure suggests that it offers little resistance to radicle emergence. The present study revealed that the endosperm in seeds of fenugreek, carob, Chinese senna, coffee and caraway has a very similar localized modification. Although the majority of the endosperm cells have thick cell walls, those in the micropylar region of the endosperm do not.

There is a transition from thick-walled to thin-walled cells between the lateral and the micropylar endosperm that appears to be dependent on the cellular position relative to the radicle tip. This defines the ‘endosperm cap’ (micropylar endosperm), which disintegrates following penetration by the radicle.

In contrast to the above, in date seeds the majority of the cells in the micropylar endosperm that surround the radicle are thick-walled. A ring of thinner-walled cells, however, exists in this region underlying the furrow that defines the operculum of the testa. Germination is completed by penetration of the radicle through the operculum, and a flap of micropylar endosperm tissue and surrounding testa, is pushed out. The presence of an operculum has been observed in other members of the palm family, e.g. Sabal minor ‘Louisiana’ (blue palm) and Zingiberales (ginger and relatives). These seeds often have a plug-like structure in the micropylar area which becomes displaced during radicle emergence by circumscissile dehiscence (Hussey, 1958). The presence of the operculum is not a universal feature of hard-seeded monocots; asparagus, for example, possesses thinner-walled cells throughout the micropylar endosperm region (Williams et al., 2001).

With respect to the role of endo-β-mannanase, it would appear only to be involved in the mobilization of the galactomannan cell walls, the major site of storage carbohydrate, in the form of hemicellulose, after germination of these seeds.

These observations on differences in endosperm structure compare and contrast with those made for the seeds of lettuce, tomato, tobacco and Datura, where the endosperm is the constraining structure to radicle emergence. In lettuce, the outer walls of the lateral endosperm are about twice as thick as those of the micropylar endosperm (Nijsse et al., 1998), although the latter still exhibit considerable thickening. There is neither transcription nor an increase in activity of the cell-wall-degrading enzyme, endo-β-mannanase, prior to radicle emergence nor any obvious degradation of the micropylar endosperm cell walls (Halmer and Bewley, 1979; Nijsse et al., 1998; Wang et al., 2004). The means by which emergence occurs is unknown. In tomato seeds, the walls of the endosperm adjacent to the cotyledons are thicker than those adjacent to the radicle, and in both regions the cells exhibit thickened walls (Toorop et al., 2000). Changes in the cell walls occur prior to germination, coincidentally with an increase in endo-β-mannanase activity; the degradation of the micropylar endosperm cell walls to permit germination is likely to require the activities of several hydrolases, however (Bewley, 1997; Bradford et al., 2000).

In Datura ferox seeds, several layers of cells are present in the micropylar endosperm; these are smaller than those in the lateral endosperm (Sànchez et al., 1990), but there is no information on differences in cell-wall thickness. There appears to be some degradation of the micropylar endosperm cell walls prior to radicle emergence, associated with a strong increase in endo-β-mannanase activity (Sànchez and de Miguel, 1997).

Thus it appears that for germination to be completed, if the walls of the micropylar endosperm cells are thickened, there is likely to be the requirement for the participation of cell wall hydrolases, including endo-β-mannanase to permit penetration by the radicle. In the case of hard-seeded species, with very thick cell walls in the endosperm, such as the hard-seeded legumes, Chinese senna, date, caraway, asparagus (Williams et al., 2001) and, perhaps, coffee, the presence of thin cell walls in the micropylar endosperm cells, in juxtaposition to the radicle, must be important in ensuring that germination can occur. In some of these species, e.g. asparagus, carob and Chinese senna, the testa may play a role in restricting germination, but primarily by limiting water uptake.