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Welwitschia mirabilis: Changes in the Megagametophyte During Early Germination
Department of Botany, University of Pretoria, Pretoria, South Africa
Welwitschia mirabilis: Changes in the Megagametophyte During Early Germination CHRIS H. BORNMAN, jOHAN P. MARAIS') and VALERIE BUTLER2) With 5 figures Received February 16, 1976 . Accepted March 16, 1976
Summary Changes in the megagametophyte of Welwitschia mirabilis over a 48-hour period after imbibition, include the partial mobilization of protein and lipid body reserves coupled with decreases in crude protein and extractable fat and increases in free sugars and total carbohydrates as well as in respiration. In particular, in cells adjoining the developing embryonic foot-like protuberance, there is an increase in RER, ER vesiculation, mitochondria, glyoxysomes and starch-laden plastids, suggesting rapid con version of lipid body reserves to sugars.
Key words: Welwitschia, Gametophyte, Protuberance, Seed.
Introduction
Welwitschia mirabilis, endemic to the Namib Desert, is a non-angiospermous gymnosperm-like dicotyledonous plant possessing advanced features such as vessels in the xylem, compound microsporangiate strobili and parallel-veined, monocotyledon-like leaves. Information is accumulating on seedling and leaf morphology and anatomy, fine structure of the phloem and transfusion tissues, uptake and movement of water and photosynthates, and on embryogenesis (RODIN, 1953,1958 a, b; MARTENS, 1971; BUTLER, BORNMAN and EVERT, 1973 a, b, c; EVERT, BORNMAN, BUTLER and GILLILAND, 1973 a, b; BORNMAN, BOTHA and NASH, 1973), but it is only comparatively recently that attention was focused on physiological and ultrastructural aspects of the germinating seed (BUTLER, BORNMAN and GILLILAND, 1973,1974; BUTLER and BORNMAN, 1975). The seeds are naked, each containing an embryo surrounded by megagametophyte, a nutritive tissue whose origin is not dependent on male gametes participating in nuclear fusion and which is the equivalent of endosperm in angiospermous seeds and ') University of Natal, Pietermaritzburg, South Africa. 2) University of Durban-Westville, South Africa.
Z. PJlanzenphysiol. Bd. 79. S. 72-80. 1976.
Welwitschia: Changes in the megagametophyte
73
sometimes erroneously referred to as such (MARTENS and WATERKEYN, 1963). While germinating, the embryo develops a peculiar wedge-shaped protuberance reminiscent of the haustorium of the primitive tracheophyte Selagin ella. Whether or not this protuberance, called feeder by BOWER (1881), has an haustorial or mechanical function, has been the subject of another study (BUTLER, 1976). Welwitschia produces vast quantities of mostly sterile and fungus-infected seed (BORNMAN et a!., 1972) and, as seed response to the supply of water might just be the key to this plant's survival, germination obviously is of interest. This paper reports on some of the early changes which occur in the megagametophyte following imbibition.
Material and Methods The outer envelopes were removed from freshly-harvested, plump, non-infected seed units of Welwitschia mirabilis. The seeds were washed in running tap water for 2 h, rinsed with 1 % Cetavlon followed by sterile w ater and germinated aseptically on moist tissue paper on an inverted beaker at 30 DC in the dark. For electron microscopical observations tissue pieces were sampled from the megagametophyte in the vicinity of the collar zone in the dry, unimbibed seed and from similar tissue surrounding the developing protuberance, 7 hand 48 h after imbibition (see arrows Fig. 2). The tissue was prepared for viewing as reported earlier (EVERT et a!., 1973 a). For chemical analysis the whol e megagametophyte (seed minus embryo) was used. Crude protein was determined using the macro-Kjeldahl method and fats were quantitatively extracted from ground tissue with diethyl ether in a Soxhlet apparatus. Fat-extracted material was analyzed for total available carbohydrates and free sugars according to MARAIS (1969). Ionic groups were removed from the clarified sugar extract by electrodialysis (MACPHERSON, 1946) and the sugars separated by descending paper chromatography and identified with an aniline spray (MARAIS, 1966). After localizing the glucose, fructose and sucrose spots on unsprayed chromatograms by means of known reference sugars, they were eluted and quantitatively determined by a modified Nelson-Somogyi method (MARAIS, DE WIT and QurCKE, 1966). Oxygen uptake and respiratory quotients (RQ) were determined on megagametophyte tissue of nearly equal fresh weight using the direct Warburg manometric method following MALEK (1965). Embryos were quickly dissected from the seeds of each of the stages immediately prior to transferring the megagametophytes to the reaction vessels. Each treatment was replicated five times. RQ values were calculated on a per mg dry weight basis.
Results Figure 1 shows an embryo removed from a 7 h-imbibed seed and Fig. 2 the development of the foot-like protuberance (P) from the embryonic collar 41 h later. Table 1 summarizes the analytical data and Figs. 3-5 represent corresponding fine structural changes in the dry, imbibed (7 h) and partly germinated (48 h) states. The most striking feature of the megagametophyte in the dry state is its massive store of reserves. It contains less than 55 % water and approximately 30 % crude protein and 35 Ofo fat. The cells are thin-walled and much of their volume is taken up
Z. PJlanzenphysiol. Bd. 79. S. 72-80. 1976.
C. H.
74
BORNMAN,
J.
P.
MARAIS
and V.
BUTLER
Table 1: Constituent manges in the megagametophyte of Welwitschia during 48 h of germination. Constituent (values 1-6 are Ofo of dry weight)
Megagametophyte Dry 7h 48 h
Fat Crude Protein Glucose Fructose Sucrose Total Available Carbohydrates Oxygen Uptake, ,ulg-1h- 1 fresh weight 8. Respiratory Quotient
35.5 28 .5 0.4 0.3 1.6 10.7 0.06
1. 2. 3. 4. 5. 6. 7.
32.7 26.1 0.5 0.4 1.8 11.1 88 .86
25.5 21.1 0.9 0.6 1.8 18.8 280 0.79
Figs. 1-2: Embryo and seed of Welwitschia mirabilis. Fig. 1: Fully-imbibed embryo (7 h) showing hypocotyl-root axis, collar zone (CO below cotyledons and suspensor (5). Fig. 2: Section through seed at 48 h exposing megagametophyte and embedded embryo with cotyledons (C) and developing protuberance (P). Arrows pinpoint area of investigation.
z.
P/lanzenphysiol. Bd. 79. S. 72-80. 1976.
Welwitschia: Changes in the megagametophyte
75
by protein bodies (Fig. 3, PB). Lipid bodies (LB) line the plasma membrane, surrounded the protein bodies and fill the inter-protein body spaces, virtually obliterating the cytoplasm. Starch, initially, is not found in large quantities and amyloplasts (A), when present, mainly occur in cells at the megagametophyte-embryo interface. The protein body itself is densely granular and sometimes includes electron dense globoids (G) and globoid cavities (GC). It is bounded by a single membrane.
Fig. 3: Reserve materials in megagametophyte cell in dry state. Lipid bodies (LB) surround protein bodies (PB). Amyloplasts (A) appear only in cells adjoining embryo. Mitochondria (M) are sparse and unstructured, cytoplasm virtually obliterated. Note globoid (G) in globoid cavity (GC) in protein body. Z. Pflanzenphysiol. Bd. 79. S. 72-80. 1976.
76
C. H.
BORNMAN, ].
P.
MARAIS
and V.
BUTLER
Lipid bodies appear to be only partly bound and occasionally are coalesced. Mitochondria are sparse and poorly defined and nuclei appear to be necrotic. The nucleus usually is grossly distorted and its nucleoplasm consists of dense chromatin ramified by pale crystalline inclusions which disperse and disappear within 24 h on imbibition. In addition to glucose, fructose and sucrose, an oligosaccharide is present. Chromatographing between the ongm and sucrose, this oligosaccharide presumably raffinose - in fact comprises the major part of the sugar fraction. In fully-imbibed megagametophyte tissue some mobilization of both protein and
Fig. 4: State of reserve materials in megagametophyte cell, 7 h. Arrows indicate granular bodies possibly consisting of ribosomal aggregates.
z.
P/lanzenphysiol. Ed. 79. S. 72-80. 1976.
Welwitschia: Changes in the megagametophyte
77
lipid (Fig. 4) has occurred. Degradation is most rapid in cells lying close to the embryo, particularly those appressed to the collar zone. On hydration the limiting membrane of the protein body is pulled away from the reserve material from within which digestion appears to begin, ultimately giving it a fenestrated appearance. Lipid bodies decrease in number and mitochondria now appear more structured. Granular bodies (arrows) possibly consisting of ribosomal aggregates are present. Microbodies, assumed to be glyoxysomes, and originally present in the dry gametophyte proliferate greatly on imbibition.
Fig. 5: At 48 h stage protein body reserves have decreased and lipids appear more diffuse. Mitochondria are more numerous and well-defined. Cell wall is thick.
z.
PJlanzenphysiol. Bd. 79. S. 72-80. 1976.
78
C. H.
BORNMAN, ].
P.
MARAIS
and V.
BUTLER
There is a slight decrease in extractable fat and crude protein, and a concomitantly small increase in total carbohydrates and free sugars. The RQ value is higher than that expected if fat was the major metabolic substrate at this stage. Another striking feature is the development of a positively geotropic, wedge-shaped or foot-like protuberance (shown in Fig. 2 at the 48 h stage) the lower surface of which is appressed to the megagametophyte. At this stage in the cells of the megagametophyte (Fig. 5) the protein body reserves are becoming largely mobilized and the bodies themselves converge, forming vacuoles in the process. Fewer and smaller lipid bodies encircle the protein bodies and they no longer line the plasma membrane. The cytoplasm becomes granular and appears more normal. There is a great proliferation of endopla$mic reticulum (ER) and many mitochondria and starch-laden plastids appear. The ER gives rise to numerous small vesicles, some of which are in contact with the protein body membranes and some of which are found in the reserve deposits themselves. There is much evidence of dictyosome activity and the walls of the megagametophyte cells in contact with the embryonic protuberance become thick, appearing spongy and mucilaginous. At this stage cells of megagametophyte and protuberance adhere together very firmly. Fat and crude protein reserves decrease by about 10 and 7 0/0, respectively, as compared with the dry state and there is a marked increase in free sugars and total carbohydrates. The RQ value suggests that now fat predominates over carbohydrate as metabolic substrate. Discussion Prior to imbibition the 10 mm-Iong dorsiventrally compressed Welwitschia seed is dry and hard; the embryo, which is ca. 2.5 mm long, likewise is dry and brittle and does not lie directly against the megagametophyte. Presupposing episodic showers of sufficient intensity and frequency to allow adequate imbibition, the embryo will swell and become appressed to the megagametophyte. The radicle emerges within about 48 h and as it starts to elongate the protuberance develops from the collared zone of the embryo. At maturity, after seven or more days of germination, this protuberance is ca. 6 mm long. At this stage the cotyledons are withdrawn from the megagametophyte and upon exposure to light green quickly. The fact that the early stages are crucial to seedling development, appears to be borne out by the subcellular changes. Mobilization of protein body reserves, which BUTLER (1976) has shown actually to consist of a protein-carbohydrate complex, is rapid. Activation of mitochondria, proliferation of glyoxysomes and rough ER, vesiculation, and dictysome activity in the peripheral cells adjoining those of the protuberance, coupled with the dispersal of lipids, progressive vacuolation, and enhanced respiration, all point to rapid macromolecular turnover. A commonly oberserved association at the 48 h stage was that of starch-laden plastids surrounded by glyoxysomes, mitochondria, lipid bodies and layers of RER,
z.
Pjlanzenphysiol. Bd. 79. S. 72-80. 1976.
'W'elwitschia: Changes in the megagametophyte
79
suggesting the conversion of stored fats to sugars. The high fat content of the megagametophyte suggests a role in the energy supply of the developing embryo and indeed there is a substantial visual decrease in the number of lipid bodies and a loss of nearly 30 % in extractable fat within 48 h. The downward shift in RQ from 0.86 to 0.79 seems to confirm that at this stage the lipids have become the major metabolic substrates. It IS not suggested that these early changes in the semiautonomous megagametophyte of Welwitschia are unique; in fact the morphological and anatomical changes seem to follow patterns similar to those reported for other germinating seeds such as Glycine max (TOOMBS, 1967), Vi cia faba (BRIARTY, COULT and BOULTER, 1970) and Setaria lutesccns (ROST, 1972) in the angiospermae and Pseudotsuga menziesii (CHING, 1972) in the gymnosermae. What is unique, at least as far as an advanced tracheophyte is concerned, is the involvement of the foot-like protuberance. However, whether partital or additional autonomy is conferred on the nuclear-inactive cells of the megagametophyte by the nuclear-active embryo cells, awaits confirmation.
Acknowledgment
We gratefully acknowledge financial support by the South African Council for Scientific and Industrial Research.
References BORNMAN, C. H., J. A. ELSWORTHY, V. BUTLER, and C. E. J. BOTHA: Welwitschia mirabilis: observations on general habit, seed, seedling, and leaf characteristics. Madoqua Ser. II, 1, 53-66 (1972). BORNMAN, C. H., C. E. J. BOTHA, and L. J. NASH: Welwitschia mirabilis observations on movement of water and assimilates under fohn and fog conditions. Madoqua Ser. II, 2,63-68 (1973). BOWER, F. 0.: On the germination and histology of the seedling of \Velwitschia mirabilis. Quart. J. Micr. Sci. XXI, 15-30 (1881). BRIARTY, L. G., D. A. COULT, and D. BOULTER: Protein bodies of germinating seeds of Vicia /aba. J. Exp. Bot. 21, 513-524 (1970). BUTLER, V., C. H. BORNMAN, and R. F. EVERT: Welwitschia mirabilis: morphology of the seedling. Bot. Gaz. 134, 52-59 (1973 a). - - - Welwitschia mirabilis: vascularization of four-week-old seedling. Bot. Gaz. 134, 59-63 (1973 b). - - - Welwitschia mirabilis: vascularization of one-year-old seedling. Bot. Gaz. 134, 63-73 (1973 c). BUTLER, V., C. H. BORNMAN, and M. G. GILLILAND: The feeder of Welwitschia mirabilis embryos. Proc. Electron Microsc. Soc. S. A. 3, 55-56 (1973). - - - Megagametophyte of Welwitschia mirabilis Proc. Electron Microsc. Soc. S. A. 4, 43-44 (1974). BUTLER, V., and C. H. BORNMAN: Nuclear and cytoplasmic inclusions in Welwitschia mirabilis. Proc. Electron Microsc. Soc. S. A. 5, 43-44 (1975).
z.
P/lanzenphysiol. Ed. 79. S. 72-80. 1976.
80
C. H. BORNMAN,
J P. MARAIS and V. BUTLER
BUTLER, Valerie: Ultrastructure of the germinating Welwitschia mirabilis seed. Ph. D Thesis, University of Natal, Pietermaritzburg, South Africa (1976). CHING, T. M.: Metabolism of germinating seeds. In: T. T. KOZLOWSKI (Ed.), Seed Biology. pp. 103-218. Academic Press, New York, 1972. EVERT, R . F., C. H. BORNMAN, V. BUTLER, and M. G. GILLILAND: Structure and development of the sieve-cell protoplast in leaf veins of Welwitschia . Protoplasma 76, 1-21 (1973 a). - - - - Structure and development of sieve areas in leaf veins of Welwitschia. Protoplasma 76, 23-24 (1973 b). MAC PHERSON, H. T.: The basic amino content of proteins. Biochem. H. 40, 470-481 (1946). MALEK, J: MeBmethoden mit dem Warburgapparat. In: A. KLEINZELLER (Ed.), Manometrische Methoden. pp. 71-115. VEB Gustav Fischer Verlag, Jena, 1965. MARAIS, J P., J L. DE WIT, and G. V. QUlCKE: A critical examination of the NelsonSomogyi method for the determination of reducing sugars. Analyt. Biochem. 15, 373-381 (1966). MARAIS, J P.: An improved method for the preparation of two spray reagents for the detection of sugars on chromatograms. S. Afr. J. Agr. Sci. 9,267-268 (1966). - The Weinmann method for the determination of total available carbohydrates in plant material containing starch. Agroplantae 1, 47-50 (1969). MARTENS, P.: Les Gnetophytes. Gebriider Borntraeger, Berlin, 1971. MARTENS, P., and L. WATERKEYN: Stem apex of Welwitschia. Phytomorph. 13, 359-363 (1963). RODIN, R. J.: Seedling morphology of Welwitschia. Amer. J. Bot. 40, 374-378 (1953). - Leaf anatomy of Welwitschia 1. Early development of the leaf. Amer. J. Bot. 45, 90-95 (1958 a). - Leaf anatomy of Welwitschia II. A study of mature leaves. Amer. J Bot. 45, 96-103 (1958 b). ROST, T . H.: The ultrastructure and physiology of protein bodies and lipids from hydrated dormant and nondormant embryos of Setaria lutescens (Gramineae). Amer. J. Bot. 59, 607- 616 (1972). TOOMBS, M. P.: Protein bodies of the soybean. Plant Physiol. 42, 797-811 (1967).
Prof. Dr. CHRIS H. BORNMAN, Department of Botany, University of Pretoria, Pretoria 0002, South Africa.
Z. P/lanzenphysiol. Ed. 79. S. 72-80. 1976.
Welwitschia mirabilis: Changes in the Megagametophyte During Early Germination CHRIS H. BORNMAN, jOHAN P. MARAIS') and VALERIE BUTLER2) With 5 figures Received February 16, 1976 . Accepted March 16, 1976
Summary Changes in the megagametophyte of Welwitschia mirabilis over a 48-hour period after imbibition, include the partial mobilization of protein and lipid body reserves coupled with decreases in crude protein and extractable fat and increases in free sugars and total carbohydrates as well as in respiration. In particular, in cells adjoining the developing embryonic foot-like protuberance, there is an increase in RER, ER vesiculation, mitochondria, glyoxysomes and starch-laden plastids, suggesting rapid con version of lipid body reserves to sugars.
Key words: Welwitschia, Gametophyte, Protuberance, Seed.
Introduction
Welwitschia mirabilis, endemic to the Namib Desert, is a non-angiospermous gymnosperm-like dicotyledonous plant possessing advanced features such as vessels in the xylem, compound microsporangiate strobili and parallel-veined, monocotyledon-like leaves. Information is accumulating on seedling and leaf morphology and anatomy, fine structure of the phloem and transfusion tissues, uptake and movement of water and photosynthates, and on embryogenesis (RODIN, 1953,1958 a, b; MARTENS, 1971; BUTLER, BORNMAN and EVERT, 1973 a, b, c; EVERT, BORNMAN, BUTLER and GILLILAND, 1973 a, b; BORNMAN, BOTHA and NASH, 1973), but it is only comparatively recently that attention was focused on physiological and ultrastructural aspects of the germinating seed (BUTLER, BORNMAN and GILLILAND, 1973,1974; BUTLER and BORNMAN, 1975). The seeds are naked, each containing an embryo surrounded by megagametophyte, a nutritive tissue whose origin is not dependent on male gametes participating in nuclear fusion and which is the equivalent of endosperm in angiospermous seeds and ') University of Natal, Pietermaritzburg, South Africa. 2) University of Durban-Westville, South Africa.
Z. PJlanzenphysiol. Bd. 79. S. 72-80. 1976.
Welwitschia: Changes in the megagametophyte
73
sometimes erroneously referred to as such (MARTENS and WATERKEYN, 1963). While germinating, the embryo develops a peculiar wedge-shaped protuberance reminiscent of the haustorium of the primitive tracheophyte Selagin ella. Whether or not this protuberance, called feeder by BOWER (1881), has an haustorial or mechanical function, has been the subject of another study (BUTLER, 1976). Welwitschia produces vast quantities of mostly sterile and fungus-infected seed (BORNMAN et a!., 1972) and, as seed response to the supply of water might just be the key to this plant's survival, germination obviously is of interest. This paper reports on some of the early changes which occur in the megagametophyte following imbibition.
Material and Methods The outer envelopes were removed from freshly-harvested, plump, non-infected seed units of Welwitschia mirabilis. The seeds were washed in running tap water for 2 h, rinsed with 1 % Cetavlon followed by sterile w ater and germinated aseptically on moist tissue paper on an inverted beaker at 30 DC in the dark. For electron microscopical observations tissue pieces were sampled from the megagametophyte in the vicinity of the collar zone in the dry, unimbibed seed and from similar tissue surrounding the developing protuberance, 7 hand 48 h after imbibition (see arrows Fig. 2). The tissue was prepared for viewing as reported earlier (EVERT et a!., 1973 a). For chemical analysis the whol e megagametophyte (seed minus embryo) was used. Crude protein was determined using the macro-Kjeldahl method and fats were quantitatively extracted from ground tissue with diethyl ether in a Soxhlet apparatus. Fat-extracted material was analyzed for total available carbohydrates and free sugars according to MARAIS (1969). Ionic groups were removed from the clarified sugar extract by electrodialysis (MACPHERSON, 1946) and the sugars separated by descending paper chromatography and identified with an aniline spray (MARAIS, 1966). After localizing the glucose, fructose and sucrose spots on unsprayed chromatograms by means of known reference sugars, they were eluted and quantitatively determined by a modified Nelson-Somogyi method (MARAIS, DE WIT and QurCKE, 1966). Oxygen uptake and respiratory quotients (RQ) were determined on megagametophyte tissue of nearly equal fresh weight using the direct Warburg manometric method following MALEK (1965). Embryos were quickly dissected from the seeds of each of the stages immediately prior to transferring the megagametophytes to the reaction vessels. Each treatment was replicated five times. RQ values were calculated on a per mg dry weight basis.
Results Figure 1 shows an embryo removed from a 7 h-imbibed seed and Fig. 2 the development of the foot-like protuberance (P) from the embryonic collar 41 h later. Table 1 summarizes the analytical data and Figs. 3-5 represent corresponding fine structural changes in the dry, imbibed (7 h) and partly germinated (48 h) states. The most striking feature of the megagametophyte in the dry state is its massive store of reserves. It contains less than 55 % water and approximately 30 % crude protein and 35 Ofo fat. The cells are thin-walled and much of their volume is taken up
Z. PJlanzenphysiol. Bd. 79. S. 72-80. 1976.
C. H.
74
BORNMAN,
J.
P.
MARAIS
and V.
BUTLER
Table 1: Constituent manges in the megagametophyte of Welwitschia during 48 h of germination. Constituent (values 1-6 are Ofo of dry weight)
Megagametophyte Dry 7h 48 h
Fat Crude Protein Glucose Fructose Sucrose Total Available Carbohydrates Oxygen Uptake, ,ulg-1h- 1 fresh weight 8. Respiratory Quotient
35.5 28 .5 0.4 0.3 1.6 10.7 0.06
1. 2. 3. 4. 5. 6. 7.
32.7 26.1 0.5 0.4 1.8 11.1 88 .86
25.5 21.1 0.9 0.6 1.8 18.8 280 0.79
Figs. 1-2: Embryo and seed of Welwitschia mirabilis. Fig. 1: Fully-imbibed embryo (7 h) showing hypocotyl-root axis, collar zone (CO below cotyledons and suspensor (5). Fig. 2: Section through seed at 48 h exposing megagametophyte and embedded embryo with cotyledons (C) and developing protuberance (P). Arrows pinpoint area of investigation.
z.
P/lanzenphysiol. Bd. 79. S. 72-80. 1976.
Welwitschia: Changes in the megagametophyte
75
by protein bodies (Fig. 3, PB). Lipid bodies (LB) line the plasma membrane, surrounded the protein bodies and fill the inter-protein body spaces, virtually obliterating the cytoplasm. Starch, initially, is not found in large quantities and amyloplasts (A), when present, mainly occur in cells at the megagametophyte-embryo interface. The protein body itself is densely granular and sometimes includes electron dense globoids (G) and globoid cavities (GC). It is bounded by a single membrane.
Fig. 3: Reserve materials in megagametophyte cell in dry state. Lipid bodies (LB) surround protein bodies (PB). Amyloplasts (A) appear only in cells adjoining embryo. Mitochondria (M) are sparse and unstructured, cytoplasm virtually obliterated. Note globoid (G) in globoid cavity (GC) in protein body. Z. Pflanzenphysiol. Bd. 79. S. 72-80. 1976.
76
C. H.
BORNMAN, ].
P.
MARAIS
and V.
BUTLER
Lipid bodies appear to be only partly bound and occasionally are coalesced. Mitochondria are sparse and poorly defined and nuclei appear to be necrotic. The nucleus usually is grossly distorted and its nucleoplasm consists of dense chromatin ramified by pale crystalline inclusions which disperse and disappear within 24 h on imbibition. In addition to glucose, fructose and sucrose, an oligosaccharide is present. Chromatographing between the ongm and sucrose, this oligosaccharide presumably raffinose - in fact comprises the major part of the sugar fraction. In fully-imbibed megagametophyte tissue some mobilization of both protein and
Fig. 4: State of reserve materials in megagametophyte cell, 7 h. Arrows indicate granular bodies possibly consisting of ribosomal aggregates.
z.
P/lanzenphysiol. Ed. 79. S. 72-80. 1976.
Welwitschia: Changes in the megagametophyte
77
lipid (Fig. 4) has occurred. Degradation is most rapid in cells lying close to the embryo, particularly those appressed to the collar zone. On hydration the limiting membrane of the protein body is pulled away from the reserve material from within which digestion appears to begin, ultimately giving it a fenestrated appearance. Lipid bodies decrease in number and mitochondria now appear more structured. Granular bodies (arrows) possibly consisting of ribosomal aggregates are present. Microbodies, assumed to be glyoxysomes, and originally present in the dry gametophyte proliferate greatly on imbibition.
Fig. 5: At 48 h stage protein body reserves have decreased and lipids appear more diffuse. Mitochondria are more numerous and well-defined. Cell wall is thick.
z.
PJlanzenphysiol. Bd. 79. S. 72-80. 1976.
78
C. H.
BORNMAN, ].
P.
MARAIS
and V.
BUTLER
There is a slight decrease in extractable fat and crude protein, and a concomitantly small increase in total carbohydrates and free sugars. The RQ value is higher than that expected if fat was the major metabolic substrate at this stage. Another striking feature is the development of a positively geotropic, wedge-shaped or foot-like protuberance (shown in Fig. 2 at the 48 h stage) the lower surface of which is appressed to the megagametophyte. At this stage in the cells of the megagametophyte (Fig. 5) the protein body reserves are becoming largely mobilized and the bodies themselves converge, forming vacuoles in the process. Fewer and smaller lipid bodies encircle the protein bodies and they no longer line the plasma membrane. The cytoplasm becomes granular and appears more normal. There is a great proliferation of endopla$mic reticulum (ER) and many mitochondria and starch-laden plastids appear. The ER gives rise to numerous small vesicles, some of which are in contact with the protein body membranes and some of which are found in the reserve deposits themselves. There is much evidence of dictyosome activity and the walls of the megagametophyte cells in contact with the embryonic protuberance become thick, appearing spongy and mucilaginous. At this stage cells of megagametophyte and protuberance adhere together very firmly. Fat and crude protein reserves decrease by about 10 and 7 0/0, respectively, as compared with the dry state and there is a marked increase in free sugars and total carbohydrates. The RQ value suggests that now fat predominates over carbohydrate as metabolic substrate. Discussion Prior to imbibition the 10 mm-Iong dorsiventrally compressed Welwitschia seed is dry and hard; the embryo, which is ca. 2.5 mm long, likewise is dry and brittle and does not lie directly against the megagametophyte. Presupposing episodic showers of sufficient intensity and frequency to allow adequate imbibition, the embryo will swell and become appressed to the megagametophyte. The radicle emerges within about 48 h and as it starts to elongate the protuberance develops from the collared zone of the embryo. At maturity, after seven or more days of germination, this protuberance is ca. 6 mm long. At this stage the cotyledons are withdrawn from the megagametophyte and upon exposure to light green quickly. The fact that the early stages are crucial to seedling development, appears to be borne out by the subcellular changes. Mobilization of protein body reserves, which BUTLER (1976) has shown actually to consist of a protein-carbohydrate complex, is rapid. Activation of mitochondria, proliferation of glyoxysomes and rough ER, vesiculation, and dictysome activity in the peripheral cells adjoining those of the protuberance, coupled with the dispersal of lipids, progressive vacuolation, and enhanced respiration, all point to rapid macromolecular turnover. A commonly oberserved association at the 48 h stage was that of starch-laden plastids surrounded by glyoxysomes, mitochondria, lipid bodies and layers of RER,
z.
Pjlanzenphysiol. Bd. 79. S. 72-80. 1976.
'W'elwitschia: Changes in the megagametophyte
79
suggesting the conversion of stored fats to sugars. The high fat content of the megagametophyte suggests a role in the energy supply of the developing embryo and indeed there is a substantial visual decrease in the number of lipid bodies and a loss of nearly 30 % in extractable fat within 48 h. The downward shift in RQ from 0.86 to 0.79 seems to confirm that at this stage the lipids have become the major metabolic substrates. It IS not suggested that these early changes in the semiautonomous megagametophyte of Welwitschia are unique; in fact the morphological and anatomical changes seem to follow patterns similar to those reported for other germinating seeds such as Glycine max (TOOMBS, 1967), Vi cia faba (BRIARTY, COULT and BOULTER, 1970) and Setaria lutesccns (ROST, 1972) in the angiospermae and Pseudotsuga menziesii (CHING, 1972) in the gymnosermae. What is unique, at least as far as an advanced tracheophyte is concerned, is the involvement of the foot-like protuberance. However, whether partital or additional autonomy is conferred on the nuclear-inactive cells of the megagametophyte by the nuclear-active embryo cells, awaits confirmation.
Acknowledgment
We gratefully acknowledge financial support by the South African Council for Scientific and Industrial Research.
References BORNMAN, C. H., J. A. ELSWORTHY, V. BUTLER, and C. E. J. BOTHA: Welwitschia mirabilis: observations on general habit, seed, seedling, and leaf characteristics. Madoqua Ser. II, 1, 53-66 (1972). BORNMAN, C. H., C. E. J. BOTHA, and L. J. NASH: Welwitschia mirabilis observations on movement of water and assimilates under fohn and fog conditions. Madoqua Ser. II, 2,63-68 (1973). BOWER, F. 0.: On the germination and histology of the seedling of \Velwitschia mirabilis. Quart. J. Micr. Sci. XXI, 15-30 (1881). BRIARTY, L. G., D. A. COULT, and D. BOULTER: Protein bodies of germinating seeds of Vicia /aba. J. Exp. Bot. 21, 513-524 (1970). BUTLER, V., C. H. BORNMAN, and R. F. EVERT: Welwitschia mirabilis: morphology of the seedling. Bot. Gaz. 134, 52-59 (1973 a). - - - Welwitschia mirabilis: vascularization of four-week-old seedling. Bot. Gaz. 134, 59-63 (1973 b). - - - Welwitschia mirabilis: vascularization of one-year-old seedling. Bot. Gaz. 134, 63-73 (1973 c). BUTLER, V., C. H. BORNMAN, and M. G. GILLILAND: The feeder of Welwitschia mirabilis embryos. Proc. Electron Microsc. Soc. S. A. 3, 55-56 (1973). - - - Megagametophyte of Welwitschia mirabilis Proc. Electron Microsc. Soc. S. A. 4, 43-44 (1974). BUTLER, V., and C. H. BORNMAN: Nuclear and cytoplasmic inclusions in Welwitschia mirabilis. Proc. Electron Microsc. Soc. S. A. 5, 43-44 (1975).
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Prof. Dr. CHRIS H. BORNMAN, Department of Botany, University of Pretoria, Pretoria 0002, South Africa.
Z. P/lanzenphysiol. Ed. 79. S. 72-80. 1976.