Cellular Aspects of Development:

Germination and Cell Division

Ferns provide experimental material for the study of cellular processes of funda-
mental significance in the development not only of ferns but also of seed plants
and bryophytes. To the long-established investigations of photomorphogenesis in
gametophytes have recently been added studies of spore germination, cell division
and cell differentiation.

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 Proceedings of the Royal Society of Edinburgh, 86B, 195-202, 1985

Mobilisation of storage reserves during fern spore germination

A. E. DeMaggio
Department of Biological Sciences, Dartmouth College, Hanover,
New Hampshire, U.S.A.
and
D. A. Stetler
Department of Biology, Virginia Polytechnic Institute and State University,
Blacksburg, Virginia, U.S.A.

Synopsis
We have studied morphological and biochemical aspects of storage reserves and their degradation in fern
spores during the germination process. The results presented here are concerned with the fate of lipid,
protein and phytic acid. Depletion of lipid reserves and breakdown of lipid bodies was directly correlated
with increased activity of glyoxylate cycle enzymes during early stages of germination. Degradation of
protein reserves coincided with the depletion of salt soluble proteins (globulins) from the spores and was
related to the time when high activity of aminopeptidase and carboxypeptidase was observed. Lipid and
protein appeared to play an important role in germination. Phytic acid reserves were present in the spore
and were hydrolysed by phytase after germination occurred. This storage material was implicated in
growth of the prothallus.

Introduction

The primary importance of storage reserves and their hydrolysis in early stages of fern
spore germination is well documented (Raghavan 1980). However, the processes
involved during mobilisation and transformation of most reserve material and their
hydrolysis products are unknown. No information is presently available to indicate
how the various storage materials are synthesised and packaged in cellular compart-
ments during sporogenesis. Moreover, only limited data are available detailing the
chemistry of the storage materials and their distribution in spores of various ferns. In
addition, few hydrolytic enzymes have been identified or biochemically characterised
in fern spores and knowledge of their activity during pre- and post-germination
events is still incomplete. The obvious absence of biochemical details during fern
spore germination prompted us several years ago to initiate a series of investigations
of the reserve materials in both chlorophyllous spores (Onoclea sensibilis and
Matteuccia struthiopteris) and non-chlorophyllous spores (Dryopterisfilix-mas).Our
initial intention was to characterise the ultrastructural patterns of storage organelles
and their breakdown and correlate these morphological features with quantative
changes in storage reserves. This preliminary work quickly led to more detailed

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 196 A. E. DeMaggio and D. A. Stetler
analyses of the storage materials and the enzymes involved during their breakdown.
Consideration is currently being given to studies of the metabolic pathways that are
active during spore germination. In this article, we present only a brief summary of
some of the work we have done on lipid, protein and phytic acid reserves.

Lipid

The spores examined contained substantial lipid reserves. In Onoclea and

Matteuccia, lipid constituted 27% of the spore weight, while in Dryopteris, approxi-
mately 40% of the spore was lipid. Lipid reserves were a prominent feature of the
cytoplasm of spores of all three species. Lipid bodies, morphologically similar to
those observed in endosperm and cotyledons of fat storing seeds (Gruber et al. 1970),
packed the cytoplasm and were closely associated with protein bodies. During
imbibition, lipids were hydrolysed and at germination the content had decreased by
12-5% in Onoclea and Matteuccia and almost 50% in Dryopteris. A similar pattern of
decrease in lipid during fern spore germination had been reported earlier (see
Raghavan 1980) and it was generally concluded that breakdown of lipids could
provide the spore with energy and materials for early cell development. However, no
information was available to indicate how lipids were metabolised or what pathways
were involved in their degradation.
From our earlier work on prothallial development in Dryopteris (Stetler and
DeMaggio 1972), we were aware of the presence of microbodies in the spore
cytoplasm. They had been described in other spores (Olsen and Gullvag 1973; Fraser
and Smith 1974) but their close association with lipid bodies in our electron
micrographs led us to suspect that they were glyoxysomes. These well-characterised
organelles contain all of the enzymes necessary to convert fatty acids to succinate
which then can be converted to sucrose. Their role in gluconeogenesis in fatty seeds is
well known. We have now established that a functional glyoxylate cycle operates in
fern spore germination during the time when storage lipids are degraded and
mobilised (DeMaggio et al. 1979, 1980). Unique glyoxylate cycle enzymes, isocitrate
lyase and malate synthase, were identified in spores of Onoclea, Matteuccia and
Dryopteris and changes in enzyme activity were followed during the germination
process. Figure 1 shows that activity of both enzymes in Onoclea increased sharply,
peaked between 4-5 days and then proceeded to decrease rapidly. The increased
activity took place as lipid reserves were being reduced and spores were undergoing
internal differentiation prior to spore coat breakage. Maximum activity coincided
with spore germination. Essentially similar results were observed by us using
Matteuccia and Dryopteris spores and by Gemmrich (1979, 1980) using spores of
Anemia phyllitidis and Pteris vittata.
These data indicate that in fern spores, as in fatty seeds, the glyoxylate cycle
operates in the conversion of storage triglycerides to sucrose. However, to demon-
strate convincingly that it represents a significant pathway for lipid catabolism
requires that the path of carbon from labelled acetate into metabolic intermediates be
traced. This work has not been completed and, therefore, we cannot rule out the
possibility that the reserve lipid is degraded in the citric acid cycle. Nevertheless, the

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 TO
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Figure 1. Time course of the changes in isocitrate lyase (ILA) and malate synthase (MS) activity during
9

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germination of fern spores, (a) activity per spore, (b) activity per mg protein.

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 198 A. E. DeMaggio and D. A. Stetler
finding that glyoxylate enzymes are active in fern spores, when coupled with recent
measurements of respiratory activity and sucrose accumulation during germination,
provides strong support for the view that, in these spores, fats are metabolised via the
glyoxylate cycle.

Protein

Proteins have been found to be an important storage material in spores of a variety

of ferns. In our studies, measurements of total extracted protein from spores of
Onoclea, Matteuccia and Dryopteris gave values of 14%, 15%, and 5% respectively.
During imbibition and germination, the protein content decreased significantly,
attaining a steady state that coincided with the emergence of rhizoid and prothallial
cell. During this time, there was an 80% reduction in storage protein of the spore.
Other investigators have noted a high protein content in ungerminated fern spores
and they also have observed an early decrease in protein as the spore germinates
(Raghaven 1980; Bassel et al. 1981). Gantt and Arnott (1965) observed that in
Matteuccia spores, protein granules decreased in number, became irregular in size
and shape, and were being digested after 12 hours incubation in the light. The results
led them to postulate that proteins were 'the primary storage products.' Similar views
of the importance of protein in early stages of fern spore germination have been
expressed recently (Raghavan 1980).
Hydrolysis of storage proteins to peptides and constituent amino acids provides the
spore with the building blocks to synthesise new proteins for the biogenesis of mem-
branes and organelles and for the production of differentiated cells of the gameto-
phyte. A great deal is known about this important process as it occurs in seeds of
higher plants but we have only begun to investigate the process in fern spores. In
Matteuccia spores, we studied in detail the structural aspects of protein body
degradation during imbibition and germination. Our electron micrographs showed
that protein bodies containing amorphous storage material and, surrounded by
smaller lipid bodies, filled the outer cytoplasm of the spore (Plate 1A). The protein
bodies were remarkably similar in their size and distribution to those described in
Onoclea sensibilis (Bassel et al. 1981). They were bordered by a limiting membrane
and began to show evidence of degradation as early as 12 hours after imbibition. As
protein was hydrolysed, there was a conspicuous decrease in the contents of the
storage bodies. After 48 hours incubation, protein body breakdown was charac-
terized by irregularly shaped organelles, diminished protein content and fusion of the
vesicles as material disappeared from the organelles (Plate IB, C).
To complement the morphological analyses of protein catabolism, we initiated
biochemical studies of the storage protein and associated hydrolytic enzymes from
Matteuccia spores. (A. E. DeMaggio, T. Templeman and H. Cohen, unpublished).
These studies have already provided some important insights into the chemical
characteristics of the storage material. Templeman has found that approximately 25%
of the total extractable protein is of the globulin type (salt soluble). Globulin protein
is known to be the principal reserve protein of a number of seeds. In the fern spore,
the globulin protein fraction decreased during imbibition and germination and much
of it had disappeared by the stage of rhizoid and prothallial formation. Electro-
phoretic analyses of this protein fraction on SDS gels revealed a number of poly-

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 Storage reserves during spore germination 199
peptide bands, many of which also disappeared from the spore during imbibition
and germination. These studies are continuing but the information they have already
yielded supports the thesis that spore storage proteins are globulins.
We also have information about the enzymatic mechanisms that are active during
the period of protein breakdown. Cohen has shown that protein degradation in the
spore is accompanied by a substantial increase in levels of amino acids. A search for
the proteolytic enzymes that were involved during the time that spore protein levels
were declining and protein bodies were degrading disclosed both aminopeptidase and
carboxypeptidase activity. Measureable aminopeptidase activity was present in the
dry spore and activity increased during the first 12 hours of imbibition before
declining to 45% of peak activity after 96 hours. He also found that carboxypeptidase
was present in the dry spore and its activity remained constant for the first 12 hours
and then declined. The close correlation between activity of these enzymes and
hydrolysis of storage protein suggests that they are an important part of the
proteolytic mechanism without which the biosynthetic events involved in germination
and prothallial development could not take place.

Phytic acid

During our ultrastructural investigations of the protein reserves in Matteuccia

spores, we observed discrete inclusions in the matrix of the proteinaceous material.
These numerous, dense particles were randomly distributed throughout the organelle
and were morphologically indistinguishable from phytic acid globoids that had been
identified and characterised in storage tissue of various seeds (Lott 1978). Plate ID
shows spore protein bodies that had not been degraded containing conspicuous
globoid inclusions. Globoids of phytic acid have been studied in protein bodies from
endosperm and cotyledonary tissue and both in vivo and in vitro analyses show them
to consist of inositol, phosphorus and various mono- and divalent cations (K + ,
Ca + + , Mg + + ). Chemically, phytic acid is known to be myo-inositol 1,2,3,4,5,6-
hexakis (dihydrogen phosphate). The compound complexes readily with cations,
although the cation content varies with seed type and tissue position (Dwarte and
Ashford 1982). It accumulates in seeds during the ripening process as other storage
reserves are being synthesised and deposited. During seed germination and early
seedling development, it is broken down by the enzyme phytase and is gradually
dephosphorylated. Various roles for phytic acid have been proposed. It is generally
regarded as a primary storage reserve of phosphorus and mineral elements (Reddy et
al. 1982). However, recent investigations have stressed its importance as a source of
inositol which can be utilised in the synthesis of cell wall components (Loewus 1983).
It is obvious that phytic acid is a primary storage material in seeds. Encouraged
by our ultrastructural evidence of its presence in spores, we attempted to identify it
chemically and determine its fate during the germination process. Here we can only
briefly summarize results of this investigation (A. E. DeMaggio and J. Soldatis,
unpublished). Dry spores of Matteuccia were extracted and assayed for phytic acid
using conventional chemical techniques (Reddy et al. 1982) and found to contain
approximately 004 mg phytic acid/gram spore. The amount did not change until the
fourth or fifth day after spores were planted in water, then it began to decline. At that

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 200 A. E. DeMaggio and D. A. Stetler
time, spores had already germinated, the spore coat was broken and rhizoid and
prothallial cell had emerged. During the next few days of growth, phytic acid levels
declined steadily and when the prothallus attained the 3-celled stage, phytic acid levels
had decreased by 50%. Measurements of inorganic phosphorus in the spore over this
time period showed that there was an increase in available phosphorus as phytic acid
was being depleted. This suggested that phytic acid was being enzymatically
dephosphorylated. We then set out to isolate and identify an acid phosphatase that
would effectively hydrolyse phytic acid, i.e. phytase. The enzyjne was subsequently
isolated, partially purified and its activity monitored during spore imbibition,
germination and early growth of the prothallus. Enzyme activity in the spore was
found to increase as the phytic acid content began to decrease. The linear decrease in
phytic acid content was closely coupled to increased enzyme activity.
These results are significant in identifying an active phytin/phytase system during
spore germination and prothallial development. The value of the system in germina-
tion and development is difficult to assess based on the limited work reported here.
However, our accumulated data indicated that 20% of the spores' total phosphate
was present in phytic acid. This, of course, becomes available to the spore for
synthesis of nucleotides, phospholipids, phosphoproteins and other phosphorylated
substances that are necessary for growth and differentiation of the young prothallus.
In addition, our studies indicate that the phytin/phytase system was not active until
spore germination occurred and rhizoid and prothallial cells had formed. Because of
the obvious association between availability of phytic acid reserves and prothallial
growth, we propose that this unique reserve material may be an important ingredient
for early stages of gametophyte development.

Conclusions

It would be premature for us to generalise from the results of our studies and
attempt to formulate biochemical schemes for germination in homosporous ferns.
Nevertheless, our work has provided us with a perspective of the biochemical
complexities of the spore and we are prompted to offer some thoughts, albeit
speculative, concerning biochemical aspects of spore germination. It appears to us
likely that most fern spores probably contain a variety of reserve materials and a
complex of regulatory mechanisms that are involved in their breakdown and
utilisation. These materials and biochemical pathways are similar to those observed
in seeds since they probably represent expressions of evolutionary experiments that
took place during the evolution of the seed. We do not envisage any single reserve
material as being capable of providing the spore with materials and energy necessary
for germination and differentiation of the gametophyte. Our work suggests that
metabolic processes in the fern spore are tightly controlled and that sequential
hydrolysis of storage reserves provides necessities for particular developmental and
physiological activities. We no longer share the view that the single-celled fern spore is
simpler and more advantageous than the seed for biochemical study. We suspect that
because of the unicellular condition, it is more important for the spore, than for the
multicellular seed, to compartmentalise'function and exert rigid temporal and spatial
controls over metabolic activities.

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 Storage reserves during spore germination 201
Acknowledgments

A part of the work summarized here was supported by funds from the Committee
on Research, Dartmouth College (A.E.D.) and by the Biology Department and the
Virginia Tech Educational Foundation (D.A.S).

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 202 A. E. DeMaggio and D. A. Stetler
Plate 1. Ultrastructure of imbibing and germinating Matteuccia spores. (A) Spores at start of imbibition
(10,000 x). (B) Spores after 48 hours imbibition showing decrease in dense proteinaceous
material and increase in vesicle size (3,300 x). (C) Spore germinating showing protein deposits
have decreased and fusion of vesicles has contributed to enlargement of the vacuolar system
(1,250 x). (D) Protein bodies containing phytic acid globoids (17,000 x).

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