ultrastructure of dry viable and non-viable protea compacta embryos

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Department of Botany, University of Natal, Pietermaritzburg, South Africa Ultrastructure of Dry Viable and Non-Viable Pro tea compacta Embryos J. VAN STADEN, M. G. GILLILAND and N. A. C. BROWN With 10 figures Received January 22, 1975 Summary An investigation of Pro tea compacta seed revealed that a number of ultrastructural dif- ferences exist between viable and non-viable embryos. The most conspicuous difference is that the lipid bodies of non-viable embryos coalesced, probably as a result of the rupturing of the «membranes» that enclosed them. On the basis of the size of the globoids present, three different types of protein bodies could be distinguished both in viable and non-viable embryos. Protein bodies containing a large number of small globoids were present mainly in the vicinity of the embryonic root tip while those with a few large globoids were found predominantly in the cotyledonary cells. Introduction A considerable amount of attention has been given to the effects of environmental factors (BROWN and VAN STADEN, 1973 a) and endogenous hormones (BROWN and VAN STADEN, 1973 b) on the germination of Protea compacta seed. It is however, becoming more and more apparent that an explanation of the mechanisms that con- trol germination is lacking. Environmental factors must of necessity act on or in- fluence specific metabolic reactions, while changes in endogenous hormone levels in many cases might well be secondary reactions that have little bearing on the germina- tion process itself. In an attempt to obtain more information about the early stages of the germina- tion process, and of the mechanisms by which it is controlled, the ultrastructure and biochemistry of proteaceous seed is being investigated. The present paper reports on the ultrastructure of dry embryos of Protea compacta. Material and Methods Dry embryos of Pro tea compacta R. BR., from two different batches of seed were used in the present investigation. One batch was a year old and viable as 68 % of the seeds ger- minated under favourable conditions (BROWN and VAN STADEN, 1973 a). The other was four Z. PJlanzenphysiol. Ed. 76. S. 28-35. 1975.

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Page 1: Ultrastructure of dry viable and non-viable Protea compacta embryos

Department of Botany, University of Natal, Pietermaritzburg, South Africa

Ultrastructure of Dry Viable and Non-Viable Pro tea compacta Embryos

J. VAN STADEN, M. G. GILLILAND and N. A. C. BROWN

With 10 figures

Received January 22, 1975

Summary

An investigation of Pro tea compacta seed revealed that a number of ultrastructural dif­ferences exist between viable and non-viable embryos. The most conspicuous difference is that the lipid bodies of non-viable embryos coalesced, probably as a result of the rupturing of the «membranes» that enclosed them. On the basis of the size of the globoids present, three different types of protein bodies could be distinguished both in viable and non-viable embryos. Protein bodies containing a large number of small globoids were present mainly in the vicinity of the embryonic root tip while those with a few large globoids were found predominantly in the cotyledonary cells.

Introduction

A considerable amount of attention has been given to the effects of environmental factors (BROWN and VAN STADEN, 1973 a) and endogenous hormones (BROWN and VAN STADEN, 1973 b) on the germination of Protea compacta seed. It is however, becoming more and more apparent that an explanation of the mechanisms that con­trol germination is lacking. Environmental factors must of necessity act on or in­fluence specific metabolic reactions, while changes in endogenous hormone levels in many cases might well be secondary reactions that have little bearing on the germina­tion process itself.

In an attempt to obtain more information about the early stages of the germina­tion process, and of the mechanisms by which it is controlled, the ultrastructure and biochemistry of proteaceous seed is being investigated. The present paper reports on the ultrastructure of dry embryos of Protea compacta.

Material and Methods

Dry embryos of Pro tea compacta R. BR., from two different batches of seed were used in the present investigation. One batch was a year old and viable as 68 % of the seeds ger­minated under favourable conditions (BROWN and VAN STADEN, 1973 a). The other was four

Z. PJlanzenphysiol. Ed. 76. S. 28-35. 1975.

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Ultrastructure of Protea Embryos 29

years old and considered to be non-viable or dead as they did not germinate under similar environmental conditions.

The tips of the embryonic root axis (first mm) and rectangular pieces (1.0 X 1.5 mm) from the cotyledons of viable and non-viable seeds were removed from the embryo. The excised material was fixed at room temperature (18 DC) in 6 % glutaraldehyde buffered at pH 7.2 with 0.05 M sodium cacodylate for 6 hours and then washed three times for periods of 30 minutes each in the same buffer. It was then post fixed in 2 % osmium tetroxide, buffered as above and again washed 3 times in 0.05 M sodium cacodylate buffer. The ma­terial was dehydrated in an alcohol series, followed by propylene oxide and embedded in araldite resin. Polymerisation lasted 48 hours at 70 DC, Sections, ca. 700 A, were cut with a diamond knife on a LKB microtome and stained with uranyl acetate and lead citrate (REYNOLDS, 1963). The sections were examined using a Hitachi HU llE electron microscope at an accelerating voltage of 50 KV and photographed.

Observations and Discussion

The use of osmium tetroxide vapours is apparently the best way to ensure that no changes occur in dry embryo material during fixation (PERNER, 1965). As found in other investigations (HORNER and ARNOTT, 1966; ABDUL-BAKI and BAKER, 1973), in the present study no differences in organelles could be observed between dry seeds and seeds that had been imbibed for six hours prior to fixation. As a result dry material fixed according to the above procedure was regarded as being represen­tative of the tissue in the dry state.

The most common inclusions that could be observed in the embryos of Pro tea compacta are protein and lipid bodies. This supports the biochemical data, which showed that on a dry weight basis the embryos contain approximately 65 % protein (total nitrogen X 6.25) and about 20 % lipid (Soxhlet reflux).

Using the electron microscope the protein bodies in the embryonic root tips of viable Pro tea compacta embryos (Fig. 1) were found to be similar in appearance to those in the aleurone layer of barley (JONES, 1969; JACOBSEN et aI., 1971), and in seed of Fraxinus excelsior (VILLIERS, 1971) and Sinapis alba (WERKER and VAUGHAN, 1974). The protein bodies vary considerably in size; from 0.5-5.0 !lm in diameter in the root tip to as much as 10 !lm in diameter in the cotyledons. They are not confined to a peripheral layer, but are distributed throughout the storage tissue of the cotyledons and the cells of the root axis. While some appear homogenous in structure, particularly in the cotyledonary tissue (Fig. 2), others have inclusions, es­pecially towards the root tip. The most common inclusion in the protein bodies is a crystalline body which occurs in cavities which are surrounded by a proteinaceous matrix (Fig. 3). These crystalline bodies were originally described by PFEFFER (1872) and are referred to as globoids. It has been suggested that phytin, the calcium or magnesium salt of myo-inositol hexaphosphate, is accumulated within these tructures (PFEFFER, 1872; Poux, 1965; SOBOLEV, 1966; JACOBSEN et aI., 1971; ROST, 1972). According to JACOBSEN et aI. (1971) globoids have three distinguishing features: they show metachromatic staining (red) with toluidin blue, they are anisotropic, and they

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30 J. VAN STADEN, M. G. GILLILAND and N. A. C. BROWN

contain high levels of phosphate that can be detected by cytochemical methods. The occurrence of phosphate has been attributed to the activity of acid phosphatase (Poux, 1965; VILLIERS, 1972). In the present investigation, when the Gomori test

for acid phosphatase was applied (BERJAK and VILLIERS, 1970), the presence of phos­phate around the globoids was confirmed as a conspicuous deposit of lead was found associated with these structures. With toluidin blue the globoids turned pink. It could, however, not be established whether they were anisotropic both before or after staining. Thus, until phytin has actually been extracted and positively identified no definite conclusion can be drawn as to whether it is the main constituent of the

globoids.

The size, number and distribution of the globoids in the protein bodies of dif­ferent parts of the embryo is of considerable interest as it might indicate different physiological functions. In the cotyledons large, relatively uniform protein bodies which are very electron dense and which contain no globoids are common (Fig. 2). Some, however, have one to several globoids lying in a cavity which is referred to as the globoid cavity (Fig. 3). In the cells near the root tip the protein bodies have a mottled and spongy appearance and contain many small cavities. Each cavity in turn contains a small globoid. These small globoids have no definite size or shape and some have a branched appearance (Fig. 4). In addition to many small globoids, the protein body as a whole may simultaneously contain one or more large globoids (Fig. 5). Progressing acropetally, there is a distinct gradation of the type of protein body from those in the root tip which contain many small globoids, to the very dense type in the cotyledonary tissue, containing none or a few globoids. In the in­termediate region between the root tip and cotyledons both types of protein body may occur in the same cell (Fig. 6).

At present very little definite information is available about the function of the globoids that occur in protein bodies. However, as the protein bodies of many seeds contain hydrolytic enzymes (MATILE, 1968; YATSU and JACKS, 1968) and as it has been suggested that these bodies may function as lysosomes in intracellular digestion (MATILE, 1968; RosT, 1972); it seems logical to assume that the globoids are in­timately involved in the digestion process itself. The presence of numerous small globoids in the root tip, that is, in the tissue that is most likely to be first activated

Fig. 1: Portion of root tip cell from viable embryos with protein bodies, lipid bodies and viable nucleus. X 6,100 (Bar 1.2 ems = 2 f1m). Fig. 2: Portion of cotyledonary cell from viable embryos with protein and lipid bodies. X 7,800 (Bar 1.6 ems = 2.um). Fig. 3: Protein bodies containing globoids in cotyledons. X 3,000 (Bar 1.2 ems = 4 ,urn). Fig. 4: Protein body with spongy texture and a large number of small globoids from root tip. X 13,000 (Bar 1.2 ems = 1 ,urn). Fig. 5: Protein body with spongy texture containing both small and large globoids in the root tip. X 20,000 (Bar 1.0 em = 0.5 ,urn). Fig. 6: Protein bodies in the root tip showing varying degrees of sponginess. X 10,000 (Bar 1 em = 1 ,urn).

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Ultrastructure of Pro tea Embryos 31

Key to lettering in figures: CLB, coalesced lipid bodies; ER, endoplasmic reticulum; G, globoid; GC, globoid cavity; LB, lipid body; M, mitochondrion; NN, necrotic nucleus; P, plastid; PB, protein body; PCB, protein carbohydrate body.

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32 J. VAN STADEN, M. G. GILLILAND and N. A. C. BROWN

upon imbibition, compared to few large globoids in the protein bodies in the coty­ledonary cells, suggests that the different globoids could have different functions. Alternatively the larger number of smaller globoids could simply serve as a means of increasing their surface area for accelerated digestion.

In addition to globoids, the dense protein bodies of the cotyledons also exhibited regions which were much less electron dense than the surrounding material (Fig. 7). In material that was fixed with potassium-permanganate these areas stained black with silver hexamine suggesting that they may be protein carbohydrate bodies (JACOBSEN et aI., 1971). Although these bodies were occasionally observed in root tip material they are more numerous in the cotyledons.

In viable seed, lipid is confined in spherical bodies or lipid bodies 0.5-1.5 11m in diameter. They stain with osmium and when mature are apparently confined by the outer layer of the original unit membrane (HORNER and ARNOTT, 1965; SCHWARZENBACH, 1971; REST and VAUGHAN, 1972). Lipid bodies are associated with the plasmalemma (Fig. 8) and are also arranged in close proximity to the unit mem­brane of the protein bodies forming a shell around them (Figs. 1, 2 and 4). The cytoplasm in the dry embryos is difficult to see as it is compressed into thin strands between the lipid and protein bodies. This is particularly true for the cotyledons where the protein bodies are very large, compact and dense. Occasionally however, mitochondria, plastids and endoplasmic reticulum can be seen on the periphery of the cells near the plasmalemma (Figs. 6 and 8). Sometimes these organelles offer sufficient resistance to be seen between the inclusions. The nucleus in the viable seed has a well defined nucleolus and the chromatin is very uniform in appearance (Fig. 1).

Ultrastructurally the protein bodies from viable and non-viable embryos are si­lar in appearance. However, three very important differences between the two seed lots could be observed. Firstly the nuclei of the non-viable embryos were necrotic. They possessed very small compact nucleoli and numerous dark condensed patches in the chromatin, considered to be heterochromatin and which apparently is indi­cative of a non-functional nucleus (Figs. 9 and 10) (VILLIERS, 1971). A second dif­ference that could be observed was that the protoplast in non-viable embryos was withdrawn from the cell wall and that the plasmalemma was damaged, especially in the root tips (Fig. 10). The third and most conspicuous difference between viable and non-viable embryos is that the lipid bodies of the latter have coalesced to form large masses of amorphous material. These have the same electron density as indi­vidual lipid bodies (Figs. 9 and 10). Together with the appearance of the amorphous material there was a reduction in the number of lipid bodies within the cells. This phenomenon was much more pronounced in the root tip than in the cotyledonary tissue. It would appear as if the »membrane« of the lipid bodies ruptured while these seeds were stored, and that this resulted in the contents of these organelles flowing together. Similar effects have been recorded when seed became infected with fungi during storage (ANDERSON et aI., 1970). In the present study no indications of fungal infection could be observed.

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Ultrastructure of Protea Embryos 33

Fig. 7: Protein bodies in cotyledonary cells showing protein carbohydrate bodies that arc less electron dense. Material fixed with KMn04. X 6,000 (Bar 1.2 cm = 2,um).

Fig. 8: Organelles in cells from the root tip. X 23,000 (Bar 1.2 cm = 0.5 ,urn).

Fig. 9: Cotyledon cells from a non-viable embryo. X 6,000 (Bar 1.2 cm = 2 [lm).

Fig. 10: Root tip cells from a non-viable embryo with necrotic nucleus and coalesced lipid. X 6,000 (Bar 1.2 cm = 2 ,urn).

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34 J. VAN STADEN, M. G. GILLILAND and N. A. C. BROWN

The changes that were observed in non-viable seeds of Protea compacta must un­doubtedly be related to their loss of viability. Apparently catabolic activity, which takes place even in dry dormant embryos (BRADBEER and COLMAN, 1967), must have occurred. The organelles that were most affected by this activity were the lipid bodies. At present it is not known whether the lipids were denatured. Lipid autoxida­tion resulting in the formation of free radicals with a resultant loss of viability is known to occur during seed ageing (KOOSTRA and HARRINGTON, 1969). A number of reports indicated that an accumulation of fatty acids in seeds results in a loss of germinability (ROBERTS, 1973). The significance of these metabolic changes taking place during seed deterioration is as yet not properly understood.

In subsequent papers ultrastructural and biochemical changes that occur in Pro tea compacta embryos during imbibition and germination will be reported on.

Acknowledgements

The authors are indebted to the Council for Scientific and Industrial Research Pretoria, South Africa for financial assistance and to the Electron Microscope Unit of the University of Natal, Pietermaritzburg for technical assistance.

References

ABDUL-BAKI, A. A., and J. E. BAKER: Are changes in cellular organelles or membranes related to vigor loss in seeds? Seed Sci. & Technol. 1, 89-125 (1973).

ANDERSON, J. D., J. E. BAKER, and E. K. WORTHINGTON: Ultrastructural changes of em­bryos in wheat infected with storage fungi. Plant Physiol. 46, 857-859 (1970).

BERJAK, P., and T. A. VILLIERS: Ageing in plant embryos. I. The establishment of the sequence of development and senescence in the root cap during germination. New Phytol. 69, 929-938 (1970).

BRADBEER, J. W., and B. COLMAN: Studies in seed dormancy I. The metabolism of (2_14C) acetate by chilled seeds of Corylus avellana L. New Phytol. 66, 5-15 (1967).

BROWN, N. A. c., and J. VAN STADEN: The effect of scarification, leaching, light, stratifica­tion, oxygen and applied hormones on germination of Protea compacta R. BR. and Leucadendron daphnoides MEISN. J. S. Afr. Bot. 39, 185-195 (1973 a).

- - The effect of stratification on the endogenous cytokinin levels of seed of Protea com­pacta and Leucadendroll daphnoides. Physiol. Plant 28, 388-392 (1973 b).

HORNER, H. T., and H. J. ARNOTT: A histochemical and ultrastructural study of Y UCC,1

seed proteins. Amer. J. Bot. 58, 1027-1038 (1965). - - A histochemical and ultrastructural study of pre- and post-germinated Yucca seeds.

Bot. Gaz. 127, 48-64 (1966). JACOBSEN, J. V., R. B. KNOW, and N. A. PYLIOTIS: The structure and composition of al­

eurone grains in the barley aleurone layer. Plant a 101, 189-209 (1971). JONES, R. L.: The fine structure of barley aleurone cells. Ibid. 85, 359-375 (1969). KOOSTRA, P. T., and J. F. HARRINGTON: Biochemical effects of age on membrane lipids of

Cucumis sativus L. seed. Proc. Int. Seed Test. Assoc. 34, 329-340 (1969). MATILE, P.: Aleurone vauoles as Iysosomes. Z. Pflanzenphysiol. 58, 365-368 (1968). PERNER, E.: Elektronenmikroskopische Untersuchungen an Zellen von Embryonen im Zustand

v6lliger Samenruhe. 1. Mitteilung. Die zellulare Strukturordnung in der Radicula lufttrock­ner Samen von Pisum sativum. Plant a 65, 334-357 (1965).

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Ultrastructure of Protea Embryos 35

PFEFFER, W.: Untersuchungen tiber die Proteinkorner und die Bedeutung des Asparagins beim Keimen der Samen. Jb. wiss. Bot. 8, 529-571 (1872).

Poux, N.: Localization de l'activite phosphatasique acide et des phosphates dans les grains d'aleurone. I. Grains d'aleurone renfermant a la fois globoids et crystalloides. J. Micro­scopie 4, 711-782 (1965).

REST, J. A., and J. G. VAUGHAN: The development of protein and oil bodies in the seed of Sinapis alba L. Planta 105, 245-262 (1972).

REYNOLDS, E. S.: The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell BioI. 17,208-212 (1963).

ROBERTS, E. H.: Loss of viability: Ultrastructural and physiological aspects. Seed Sci. & Technol. 1,529-545 (1973).

ROST, T. L.: The ultrastructure and physiology of protein bodies and lipid from hydrated dormant and non-dormant embryos of Setaria lutescencs (Gramineae), Amer. J. Bot. 59, 607-616 (1972).

SCHWARZENBACH, A. M.: Observations on spherosomal membranes. Cytobiologie 4, 156-147 (1971).

SOBOLEV, A. M.: State of phytin in aleurone grains of mature and germinating seeds. Fiziol. Rast. 13, 193-200 (1966).

VILLIERS, T. A.: Cytological studies in dormancy I. Embryo maturation during dormancy in Fraxinus excelsior. New Phytol. 70, 751-760 (1971).

- Cytological studies in dormancy II. Pathological ageing changes during prolonged dormancy and recovery upon dormancy release. Ibid. 71, 145-152 (1972).

WERKER, E., and J. G. VAUGHAN: Anatomical and ultrastructural changes in aleurone and myrosin cells in Sinapis alba during germination. Planta 116, 243-255 (1974).

YATSU, L. Y., and T. J. JACKS: Association of lysosomal activity with aleurone grains in plant cells. Arch. Biochem. Biophys. 124, 466-471 (1968).

J. VAN STADEN, M. G. GILLILAND and N. A. C. BROWN, Department of Botany, University of Natal, Pietermaritzburg, South Africa.

Z. PJlanzenphysiol. Ed. 76. S. 28-35. 1975.