Seed physiology: From ovule to maturing seed

T H E B O T A N I C A L R E V I E W VOL. 50 APRIL-JUNE, 1984 NO. 2

Seed Physiology: From Ovule to Maturing Seed

MICHAEL EVENARI Depar tment o f Botany, Hebrew University, Jerusalem, and

The Jacob Blaustein Institute o f Desert Research, Sde Boqer Ben Gurion University o f the Negev, Israel

Abstract .................................................................................................................................................................................... 143 Zusammenfassung ............................................................................................................................................................ 144 Introduction .......................................................................................................................................................................... 145 Ovule and Fertilization (Phase C of Fig. 1) .................................................................................................. 150 Endosperm and Embryogenesis (Phase D of Fig. 1) .............................................................................. 158

1. Developmental Patterns ............................................................................................................................. 158 2. Metabolic Processes ...................................................................................................................................... 162

Concluding Remarks ...................................................................................................................................................... 166 Acknowledgments ............................................................................................................................................................. 167 Literature Cited .................................................................................................................................................................. 167

Abstract

1) The future o f the seed is partly predetermined by events (flower formation, flowering, nutrient flow from mother plant, etc.) preceding fertilization and the format ion of the gametophyte.

2) The environmental conditions under which the seed matures affect its final physiological constitution. This faet has mostly been neglected by seed physiologists.

3) It is not known how far the triantic nature o f the diaspore (seed coat, pulp, etc., 2n o f mother plant, embryo n o l d + n of~, endosperm 2n of

+ n of ~) affects seed development and germination. 4) The integuments of the ovules o f some species have stomata. It is

not known if they are functional in gas exchange or are constitutional non-functioning relics.

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The BotanicaI Review 50: 143-170, April-June, 1984 143 �9 1984 The New York Botanical Garden

144 THE B O T A N I C A L REVIEW

5) The causes of the growth-degeneration pattern of the nucellus are unknown.

6) During the development of the megaspore mother cell into the mature embryo sac dramatic cellular ultrastructural changes take place. This probably signifies a "change of guards" during which the gametophyte is freed from part of the controls by the ultrastructural units of the mother plant, preparing the ground after fertilization for a new, genetically independent sporophyte.

7) Upon closer examination, the seemingly simple processes of fertilization and embryogenesis, as described in textbooks, turn out to be very complex and full of problems. Is the role each male nucleus plays preordained or is it left to chance which male nucleus goes where? What causes the degeneration of the synergids and of the vegetative nucleus, and what protects the other two male nuclei from a similar fate? Which ultrastruc- rural organelles are carried by the generative nuclei into their respective receptor cells and what is their role in them? Why do zygotes in some species develop after fertilization immediately into an embryo whereas in other species the zygote remains dormant for some time? What causes the polarity of the egg cell which, after fertilization, divides into one developmentally most active apical cell (giving rise to the embryo) and into another "lazy" basal cell which develops into the suspensor of "unknown function?"

8) In the source-sink relationship between photosynthesizing organs and the maturing seed there is one point at which the photosynthates pass from symplast to apoplast to symplast. The mechanism involved is largely unknown as well as the effect which environmental conditions have on this transport.

Zusammenfassung

1) Das Schicksal des Samens wird zum Teil durch Ereignisse bestimmt (Bltitenbildung, Bltihen, N~ihrstoffzufuhr von der Mutterpflanze u. s. w.), die der Befruchtung und der Bildung des Gametophyten vorausgehen.

2) Die Umweltsbedingungne, unter denen der Samen reift, beeinflussen seine physiologische Konstitution. Die meisten Samenphysiologen haben diese Tatsache nicht gentigend beachtet.

3) Es ist nicht bekannt, wie weit die triantische Natur der Diaspore (Samenschale, Perisperm etc., 2n der Mutterpflanze, n d + n d des Em- bryos, 2n 2 + n ~ des Endosperms) Samenentwicklung und Keimung beeinflussen.

4) Die Integumente der Samenanlagen haben bei manchen Pflanzen Spalt6ffnungen. Es ist unbekannt, ob diese eine Rolle im Gasaustausch spielen oder ob sie nur konstitutionelle nichtfunktionelle Relikte sind.

SEED PHYSIOLOGY: OVULE TO MATURING SEED 145

5) Die Ursachen des anfdnglichen Wachstums und der folgenden De- generation de Nuzellus sind nicht bekannt.

6) W/ihrend der Entwicklung der Embryosackmutterzelle zum Em- bryosack findet eine fundamentale ,~nderung der zellul/iren Ultrastruktur statt. Die Bedeutung dieser Anderung liegt m6glicherweise darin, dass sich dutch sie der Gametophyt yon der Kontrolle durch die ultrastrukturellen Organellen der Mutterpflanze befreit und somit die Basis fftir einen nach der Befruchtung genetiseh unabh/ingigen Sporophyt gelegt wird.

7) Bei n/iherer Betrachtung zeigt sich, dass Befruchtung und Embryoent- wicklung, die in den Lehrbtichern meist als unkomplizierte Vorg/inge dargestellt sind, ~iusserst komplexe und problematische Prozesse sind. Ist die Rolle der beiden generativen Kerne pr~idestiniert oder ist es dem Zufall tiberlassen, wohin jeder der beiden Kerne wandert? Was ist die Ursache der Degeneration der Synergiden und des vegetativen Kerns, und was schiJtzt die beiden generativen Kerne vor einem ~ihnlichen Los? Welche ultrastrukturellen Organellen wandern mit den generativen Kernen in ihre respektiven Rezeptorzellen, und welche Rolle spielen sie dort? Warum entwickeln sich in einigen Arten die Zygoten direkt nach der Befruchtung zu Embryonen w/~hrend sie in anderen Arten ftir oft lange Zeitr/iume im Ruhezustand bleiben? Was verursacht die Polarit/it der Eizelle, welche sich nach der Befruchtung in eine entwicklungsm~issig /iusserst aktive apikale Zelle, aus der sich der Embryo entwickelt, teilt, und in eine basale "tr/ige" Zelle, die sich zum Suspensor "unbekannter Funktion" entwickelt?

8) An einem bestimmten Punkt der Leitungsbahn, durch die Assimilate der photosynthetisierenden Organe in die reifenden Samen bef6rdert wer- den, passieren die Assimilate vom Symplast zum Apoplast zum Symplast. Sowohl Transportmechanismus als auch der Einfluss von Aussenbedin- gungen auf ihn sind im grossen Ganzen unbekannt.

Introduction

In our time, the legal problems concerning the permissibility of abortion and the possibility of creating test tube babies have raised one basic question which is being vehemently discussed: At what point does a new human life start in the mother's womb?

A review of seed physiology faces the exact same problem. What is the starting point of a seed's life cycle? The simplistic answer to this question is that the seed comes into being the moment that the nucleus of one of the generative or sperm cells has fused with the nucleus of the egg cell. This is true in a purely existential sense. But does seed physiology also begin only at the moment a seed's life begins? The answer is a definite "no." The seed's future is partly predetermined by events taking place

146 THE BOTANICAL REVIEW

before fertilization. The specific structure and physiology of each specific male and female gametophyte, which are partly controlled by the genome and partly by environmental parameters, determine the fate of the maturing seed and even its germination characteristics. But even events preceding the formation of the gametophytes, like flowering (its timing, environmental conditions at flower formation, position of flowers, etc.), have an impact on seed formation (the "legacy of the mother plant," Bewley and Black, 1978).

For the purpose of this paper, the formation of the female gametophyte is taken as the point of departure. This is a purely arbitrary decision since the real life history of a seed starts at stage A of Figure 1.

This figure needs some explanation. First of all, it demonstrates the simple fact that the developmental changes taking place during the seed's evolvement are accompanied by changes in the environmental conditions, which are different from phase to phase, and for each phase, from year to year. Each phase is, therefore, under the control of its own specific environmental conditions (as indicated in Fig. 1 by the different Roman numerals for the various environmental parameters), in addition to the control exerted by the genome. There is a continuous interaction between these two types of control. Through transcription and translation, each gene of the genome finds its expression in the phenotype at a certain preordained time and phase of development, under given internal and external environmental conditions which can change the phenotypic expression of the gene. As far as the internal environment is concerned, e.g. the environment of the intercarpelar cavity inside an ovary to which the ovule is exposed, there is even a gene expression-environment feedback system involved.

It is necessary to restate these well known facts because it is quite remarkable that the investigators studying the physiological-biochemical processes of phases III and IV (Fig. l) more or less completely disregard the impact of the environmental factors on these events and on the phenotypic expression of the genes active at these times. A good example of this is a statement by Sussex and Dale (1979): " . . . development from ovule to mature seed appears to be largely independent of informational cues from the maternal plant and from the external environment and is therefore primarily under genetic control." It is also probable that the

Fig. 1. Schematic drawing representing the main stages of the development of fruit (fleshy) and seed (with endosperm) in its dependence upon environmental factors. SWP-- soil water potential, N--nutrients contained in soil, PWP--plant water potential, PHS-- photosynthates, PHP -- photoperiod, THP -- thermoperiod, T- - temperature, Li-- light, FC -- fruit coat, FP--frui t pulp, SC--seed coat, EN--endosperm, E--embryo.

SEED PHYSIOLOGY: OVULE T O M A T U R I N G SEED 147


environmental parameters not only affect gene expression during these phases of seed formation, but also serve as time signals for transcription and translation of the genes involved, another environmental-gene-phenotype controlling relationship worth investigating. It is quite the other way around for phases V and VI, where much attention has been paid to the environmental impact on the events and comparatively little to their genetic control.

Another neglected effect is the genetic control of the formation of the dispersal unit (diaspore). Until the formation of the mature but not yet fertilized embryo sac, the 2n genetic complement of the mother plant controls the development as far as it is dependent on heredity. This changes with the double fertilization of the egg and polar nuclei. From then on the diaspore coat, diaspore flesh and pulp, seed coats and some storage organs like the perisperm, remain under the genetic control of the mother plant, whereas the embryo's genetic control is now exerted by the complement of the n female plus the n male, and the endosperm's genetic control by its 3n, composed of 2n of the mother plant and n of the male fertilizer. A 3n endosperm is the "normal" case but it should not be forgotten that in some plants the endosperm's polyploidy amounts to 6n, 10n, 15n and even more (Bhatnagar and Johri, 1972; Johansen, 1951). [The same authors report that the nuclei of the endospermal haustoria of Arum maculatum contain 2457n and that in Pedicularis palustris parts of the endosperm are hexaploid and others dodekaploid.]

As far as can be seen, we have no knowledge about the physiological- biochemical impact of the genetically triantic entity (from Greek, "tria," three) of the diaspore on diaspore development and seed physiology. This gap in our knowledge becomes even larger when we ask" Do these three different genomes act independently from each other when exerting their genetic control on the organs containing that specific genetic complement or do they interact when expressing themselves in the phenotype? Since the diaspore is a triantic entity in which the different parts interact during development and later germination, the assumption for genome interaction has a higher degree of probability than the assumption for non- interaction.

Another problem concerns the multi-faceted role of the mother plant in diaspore development. The mother plant provides most (sometimes all) of the energy needed for diaspore development and all of it needed for the germination of the seed which it transports and deposits into the storage organs of the diaspore. But here the question arises whether this is a one-way process or whether the developing seed has, in this respect, a feedback relationship with the mother plant. It seems probable that such a feedback exists, but very few papers touch upon this problem. This relationship between the energy-providing entity and the receiving sink


is naturally also affected by the environmental conditions under which the process takes place, another question about which we know very little.

During maturation, the diaspore becomes increasingly independent of its mother plant and at a certain developmental phase the vascular connection between mother plant and diaspore is severed. From then on all the internal control systems are located exclusively in the autonomous diaspore. This is comparable to the cutting off of the umbilical cord between mother and new-born child, a process of decisive consequences for the child. In the diaspore, the analogous event also has far reaching consequences.

In Figure 1, the thick arrows between the various phases indicate not only the sequence of events, but point out another problem: the various phases are not independent units. Each one carries in some way, the imprint of the preceding phase or phases. This again is a "common-place" statement, "common" to all processes of development and change in our continuously changing world (including human history). This "historic" effect has both a direct and an indirect implication. It is obvious that besides other environmental factors, the photo- and thermo-periodic conditions under which the plant grows during phase I will affect the flower, and thereby seed formation at phases II and III, the nutrient and water supply during phases I and II will have its impact on the number of flowers and seeds during phases III and IV, etc. These direct effects have generally been well studied though there are gaps even in our knowledge of these "transfer" processes and their action mechanisms. The indirect "historic" effects, the "legacies," are much less known.

An example of such a legacy is the fact that the environmental conditions of the mother plant at phases II to IV have an effect on the germinability of the seeds. In this case the time gap between cause and effect may be years. It may well be that we deal here not only with a purely environmental legacy, but also with an environmentally condi- tioned genetic legacy. It is quite possible, and perhaps even probable, that the synthesis of those RNA species which are formed during seed maturation but remain untranslated until the occurrence of germination, is affected by environmental conditions of the mother plant. It is as if during germination, the seeds "remember" preceding events.

Concluding these general remarks, two more points have to be made. 1) This review deals only with angiosperms since comparatively little

is known about the gymnosperm's seed physiology. 2) Most research on seed physiology is centered on a few plants, mostly

agricultural ones like wheat, barley, rice, soy-beans, maize, peas, etc. This is quite understandable since the results obtained with these crops are sometimes of considerable practical importance. But this raises the question as to what extent do the results obtained with these plants--which


have been subjected to man's selection, unconsciously and consciously, since the invention of agriculture--can be extrapolated to other non- domesticated plants.

Ovule and Fertilization (Phase C of Fig. 1)

REFERENCE REVIEWS: Bewley and Black, 1978; Bhatnagar and Johri, 1972; Fahn, 1982; Jensen, 1972; Maheshwari, 1950; Tilton, 1981; Tilton and Lersten, 1981; Wardlaw, 1955.

The change-over from the female sporophyte to the female gametophyte is accompanied by a number of events which partly determine the developmental stages that follow.

We will deal first with the maternal sporophytic tissues of the ovule surrounding the embryo sac, i.e. the integument(s), nucellus and funiculus.

The integument(s) may develop before archespore initiation; it may develop simultaneously with it or during meiosis (Fahn, 1982; Tilton and Lersten, 1981). Depending on the species, integumentary cells contain carbohydrates (starch grains), lipids and protein bodies, and apparently serve as a storage organ. Their contents change with pollination and fertilization. In Nicotiana rustica, for example, the integumentary cells contain many protein bodies which disappear after pollination when starch makes its appearance. During embryogenesis starch is catabolized and disappears (Sehgal and Gifford, 1979). In N. tabacum lipid bodies in the integumentary cells are replaced by starch after fertilization (Mogensen and Suthar, 1979). The integument(s) are covered by a cuticle (Mogensen, 1981 a; Tilton and Lersten, 1981) and in many cases, plasmodesmata are present in certain cells of the integument(s). The endothelium (integumental tapetum), which is either the inner layer (inner epidermis?) of the integument(s), which is the "normal" case, or the outer layer (outer epidermis?) of the nucellus (Kapil and Tiwani, 1978; Tilton and Lersten, 1981), plays an apparently very important physiological role in mega- sporo- and embryogenesis.

The endothelial cells are often polyploid, contain mitochondria, ribosomes, endoplasmic reticulum (ER), vacuoles, vesicles, proplastids with starch, high concentration of protein and high levels of DNA (at least in some cases) and enzymes like amylases, proteinases, etc. (Kapil and Ti- wani, 1978; Mogensen, 1981a, 1981b; Mogensen and Suthar, 1979). If, as in Nicotiana rustica, the endothelium contains little starch and protein, Sehgal and Gifford (1979) ascribe this " . . . to their quick mobilization by hydrolytic enzymes. . , and the translocation of materials to the embryo sac." All the facts point therefore to a nutritional and perhaps even a "digestive" (Guignard, 1893; Kapil and Tiwani, 1978) role of the endothelium. In addition to this nutritional function, the integument(s) also


protects the ovule against desiccation (presence of cuticle) and mechanical injury (Tilton and Lersten, 1981). It is also important because it does not cover the nucellus completely, but leaves an opening at one end, the micropyle, through which, in most cases, the pollen tube enters the nucellus. During embryogenesis the integument(s) and the endothelium undergo considerable changes. Its outer layers become thick-walled and dead, its inner walls disintegrate, processes which change its function, and the integument(s) now constitutes the seed coat with its specific new function.

Most seed physiologists have overlooked the unusual fact that in many species of various families the integuments have stomata and even chlorenchyma (first reported by Haberlandt, 1877, 1884; Bhatnagar and Johri, 1972; Corner, 1976; Fahn, 1982; Jernstedt and Clark, 1979; Netolitzky, 1926). These integumental stomata persist during embryogenesis and seed ripening, and are present in the mature seeds (Jernstedt and Clark, 1979, for Eschscholzia; Rugenstein and Lersten, 1981, for eight species of Bau- hinia). In most of these cases, it is not known at which point during the development of the integuments the stomata are formed, but for Gos- sypium, Joshi et al. (1967) report that "they appear 2 days before anthesis, and remain healthy (does that mean functional? M.E.) up to 14 to 16 days after pollination." The guard cells are gorged with starch grains (Bhatnagar and Johri, 1972). Since there is no investigation of the function of these integumental stomata it can only be guessed that they play a role in respiration and perhaps even in the photosynthesis--if chlorenchyma is present--of the ovule.

It may be strange to ascribe to them a function in photosynthesis since it seems improbable that the ovule, enclosed in the ovary, receives enough light for photosynthesis. There is only an indirect indication that photosynthesis may take place in ovules. Jernstedt and Clark (1979) reported that enough light passes through the fruit coat of Eschscholzia to enable the seeds which have stomata to photosynthesize. If this is true for mature seeds, it seems most likely that it will be true also for ovules. In this regard it would be irrelevant whether the stomata are able to open and close during the whole process of seed morphogenesis, or can do so only during megasporogenesis (case of Gossypium?) and later stay open (case of Eschscholzia), since in their position the danger of losing too much water by transpiration is most unlikely.

Our knowledge of the function and physiological causes of the odd growth-degeneration pattern of the nucellus and its hypo- and epistasis is most scanty. The development of the ovule starts with the initiation of the nucellus. In this context the question arises, "What is the stimulus which sets in motion the chain of events leading to the formation of the ovule?" Whereas the genesis of the ovary is causally a consequence of


flower formation, this cannot be said with certainty of the genesis of the ovule since there are many cases of ovaries devoid of ovules.

The nucellus, which produces the female gametophyte, stores lipids and starches which serve as nutrients for the megasporogeneous tissues (Tilton and Lersten, 1981). During megasporogenesis all or part of the nucellar cells degenerate (an autolytic process?) and their content is absorbed by the megasporophyte. Apparently the cuticle which covers the nucellus, and in many cases also the endothelium, remains. In some cases the degeneration process is accompanied by proliferation of some of the nucellar cells at the microplar and/or chalazal end of the ovule, forming epi- and/or hypostases.

The funiculus, another sporophytic part of the ovule, ties the ovule to the placenta, and through its vascular bundles, water and nutrients are transferred from the mother plant into the ovule. After its abscission from the mature seed, the funiculus leaves a legacy to the seeds: the hilum, with its cleft, and, in some species, the strophiole, which fulfill an important function in seed germination.

The most dramatic cellular ultrastructural changes take place when the megaspore mother cell (meiocyte, MMC), included in the yet undiffer- entiated ovule develops into the mature embryo sac ready for fertilization surrounded by the nucellus and the integument(s). In many cases callose is formed around the MMC either before or during meiosis, leaving the chalazal end of the meiotic cross walls completely or partly free. In other cases the callose formed around the whole MMC is later dissolved at the chalazal pole of the embryo sac mother cell (EMC). This insures transfer of solutes from nucellus to the MMC and EMC in spite of the presence of the callose (Russel, 1979; Tilton, 1980).

An even more profound change concerns the organelles of the MMC which disappear before and during meiosis. New organelles then appear in the EMC (Dickinson and Heslop-Harrison, 1977; Tilton, 1981). The case of Capsella, described by Schulz and Jensen (1968, 1974, 1981), is given here in detail as an example. Already in prophase I, differently structured cisternae appear which "encapsulate cytoplasm, ribosomes, plastids, mitochondria and ER." These particles are destroyed during meiosis. The cisternae contain acid phosphatase and are interpreted by the authors as "autophagic vacuoles that function in the destruction of substances that control the expression of the sporophyte generation during the changeover to the gametophyte phase of the life cycle." Some of the sporophytic plastids and mitochondria are apparently not destroyed and become part of the female gametophyte (embryo sac), whereas the ribosomes of the surviving megaspore are newly synthesized when new ri- bosome subunits "outpour" from the megaspore's nucleolus.

Another important fact reported by Schulz and Jensen (198 l) concerns


the existence of plasmodesmata in the walls of the MMC, indicating its communication with the nucellus for the purpose of transport of various materials, a point to which we will come back later.

Schulz and Jensen's theory concerning the destruction of control-exerting sporophytic units during embryo sac development is most attractive since it would signify a "change of guards" enabling the mature gametophyte, freed (with the exception of only a restricted number of ultrastructural sporophytic units) from the controls imposed by most of the ultrastructural units of the mother plant (which still exist for the nucellus, integuments and ovary), ready to start a new sporophytic life after fertilization and ready to give rise, after fertilization, to a new, genetically independent sporophyte.

From an evolutionary point of view, the Schulz and Jensen theory fits in well with what we have known since Hofmeister's (1851) classical work about the alternation of generations of archegoniates and spermatophytes. In his book, Hofmeister showed that the free living gametophyte of the archegoniates is homologous to the gametophyte of the spermatophytes which remains enclosed in its spermatophyte. The theory of Schulz and Jensen indicates that the gametophyte of the spermatophytes still carries some legacy of the physiological independence of the free living gametophyte of the archegoniates because its ultrastructure seems to be independent from that of the enclosing sporophyte by which it is only "hosted" like a parasite living on its host plant. The fact that callose, which cuts the material communication between MMC and nucellus, is formed around the MMC (homologous to the spore mother cells of the archegoniates) and the EMC, points also to the same conclusions.

There remain several questions concerning the development of the embryo sac which are relevant to our topic:

1) Are the results obtained in a few cases typical for all or most cases? Capsella may represent only one type [out of at least 13 according to Johri (1963)] of megagametogenesis.

2) The nutritional dependence of the megagametophyte upon the female sporophyte indicates that environmental conditions must have an indirect and a direct effect on this event: indirect, because the nutritional status of the mother plant, which in turn affects megagametogenesis, is controlled to a large extent by environmental parameters, and directly, because the same parameters must also directly affect the embryo sac development and not only through the female sporophyte. As far as can be seen from the literature this seems to be a much (or perhaps completely?) neglected problem.

The structure and function of the mature embryo sac of the angiosperms and the fertilization process are obviously of vital importance to under- standing seed formation and physiology. In this paper, only the "normal"


(term used by Jensen, 1972) type of embryo sac can be dealt with, where the term "normal" is justified only because it is the type found in most cultivated plants. This type of embryo sac is certainly not "normal" when taking into account the many species in which the embryo sac is structured differently (Maheshwari, 1950). As a point of departure for the following discussion of the function of the embryo sac and its components, a brief description of the various embryo sac cells is given (Jensen, 1972; Tilton, 1981).

The "normal" embryo sac contains the egg apparatus (one egg cell and two synergids), the central cell with its polar nuclei and the synergids (three cells). Besides the large nucleus, the egg cell contains in its cytoplasm, plastids (sometimes with starch), mitochondria, dictyosomes, rough ER and free and ER bound ribosomes and microbodies. But all these organelles are present only in relatively small numbers. The nucleus and most cytoplasm are located at the chalazal end of the egg cell and a large vacuole at its micropilar side. In some cases vacuoles are absent. Are these organelles inactive until after fertilization, as has been supposed (Jensen, 1972; Tilton, 1981), and if so, exactly when do they become active after fertilization? What event activates them and what is the activation mechanism?

The problem of activation after fertilization is of special interest since Seghal and Gifford (1979) found RNA bodies near the nucleus before fertilization in the egg cells of Nicot iana rustica. They speculate that they could have been produced by the egg genome and be of a "messenger nature," producing, after fertilization, enzymes needed for the early development of the zygote. All these questions, to which we will come back to when dealing with fertilization, are related to the fact that in many cases, the zygote remains "dormant" for hours, days or even months, as is the case, for example, in Pistacia vera where the zygote starts to develop only 10-12 weeks after fertilization (Grundwag and Fahn, 1969). The reasons for this zygote dormancy are yet unknown as are also the reasons for the termination of dormancy, i.e. the stimulus which causes the dormant zygote to develop. There may be a relationship between the zygote's behavior and the endosperm because, in many cases, the zygote's first divisions are preceded by the division of the fertilized endosperm nucleus (post hoc propter hoc?), but if so, the same question would have to be asked for the endosperm nucleus.

A unique feature of the synergids, term coined by Schacht in 1857 (the "step children" of the embryo sac, Jensen, 1972), is the filiform apparatus at their micropilar end which contains carbohydrates, proteins and RNA (Tilton, 1981). Plastids, dictyosomes, vesicles, "sphaerosome like bodies" (?), rough and smooth ER, ribosomes, mitochondria and vacuole(s) (containing carbohydrates?) are found throughout the synergids and are con-


centrated at their chalazal end. Synergids are metabolically very active and play an important role in reproduction. There are suggestions that they play some role in nutrient transfer to the embryo sac (Jensen, 1972). This has been supported by some modern research.

Bentwood and Cronshaw (1978), Sehgal and Gifford (1979) and Mo- gensen (1981a, 1981b) have shown that the synergids can fulfill such a function only because the integumental cells near the micropyle, and especially the endothelium, contain ATPase (for the role of ATPase in cells transferring solutes in connection with the sieve tubes, see Bentwood and Cronshaw, 1978) and therewith the "necessary enzymes for active (emphasis mine) translocation of solutes into the embryo sac" (Mogensen, 1981 a). These solutes are transported to ovules by the vascular bundles of the funiculus, which constitutes the communication link between ova- ties and mother plant at the one end, and ovule and embryo sac at the other. From the end cells of the vascular bundles the solutes diffuse (?) to the endothelium which secretes them into the synergids, which then, according to Mogensen (1981 a, 1981 b), function as "more passive" (Mo- gensen, 1981 a) absorption cells, a function which is apparently mediated by their convoluted filiform apparatus. Tilton (1981) thinks that the nutritional function of the synergids is still "speculative" and of "trivial consequence."

However this may be, there is a consensus of opinion that the synergids are secretory cells producing and excreting substances which direct the pollen tube chemotropically to the micropyle (Bhatnagar and Johri, 1972; Jensen, 1968b, 1972; Welk et al., 1965).

Concerning the nutrition of the embryo sac, there remains the puzzling problem of the hypostase and epistase tissues found in the nucellus of some families. As far as I can see, these organs have been completely neglected by the seed physiologist. Bhatnagar and Johri (1972) write that in addition to their probable function to "stabilize the water balance of the resting seed" (how?), they may also "check the growth of the embryo sac." The hypostase, which stores starch, is apparently involved in the nutrition of the embryo sac (Tilton, 1980; Tilton and Lersten, 1981; Ventakao Rao, 1953).

The cytoplasm of those antipodal cells which have been ultrastructurally investigated contains all organelles associated with intense metabolic activity; but their function is not clear, especially since they are ephemeral in some plants and persistent in others, and their structure varies con- siderably from plant to plant. The following quotation from Jensen (1972) is as valid today as when it was written: "This great variability in the conditions of the antipodals make it impossible to characterize this group of cells in any general manner."

The central cell ("the forgotten child," Jensen, 1972), with its two polar


nuclei, has a vacuole(s), plastids, much ER, many dictyosomes, ribosomes and microbodies. The plasma membranes of the central egg and synergid cells are in direct contact because they lack cell walls in this region. The primary endosperm nucleus formed by the fusion of the two polar nuclei with one of the male nuclei, give rise, by repeated division, to the endosperm.

When germination of pollen on the stigma, its penetration through the style to the embryo sac, fertilization and double fertilization were first discovered (see Table I, Evenari, 1984), the whole process of fertilization appeared to be a relatively simple phenomenon. The description of fertilization in the most modern botany textbooks confers the same impression upon the reader. But, in reality, the more research undertaken concerning fertilization, the more evident it becomes that one is dealing here with a most complex problem. In discussing fertilization, all the numerous problems concerning the growth of the pollen tube will be excluded. Discussion will focus on the status of the pollen tube when it has reached the embryo sac, and the process of the fusion of the two generative nuclei with the egg cell nucleus and the primary endosperm nucleus respectively.

When the growing tip of the pollen tube, which is rich in protein, RNA and carbohydrates, reaches the micropyle, the greatest part of the protoplast of the pollen tube is separated from the two generative cells by a number ofcallose plugs. In this non-growing callose plug formation zone, Golgi bodies, amyloplasts, mitochondria, ribosomes, lipid bodies, rough ER and vacuoles are found, organelles which cannot be spilled into the embryo sac (Cresti et al., 1976; Jensen, 1968b; Sanger and Jackson, 1971). At this stage the vegetative nucleus may still be in front of the two generative cells or may already have degenerated. The detection of a micro- fibrillar body with microtubules in the cytoplasm of the generative cells, in some cases, and in the pollen tube cytoplasm in others, has led to the assumption that these bodies are related to the movement of the generative cells and the vegetative nucleus (Brighigna et al., 1980, and literature cited there).

It is not very clear what exactly happens after the tips of the pollen tube have reached the micropyle of the megagametophyte, apparently directed there by the excretion of chemotropical substances by the synergids (Bhatnagar and Johri, 1972; Jensen, 1972; Jensen et al., 1977; Mo- gensen, 1978; Mogensen and Suthar, 1979; Tilton, 1981). The pollen tube enters one synergid through its filiform apparatus. At this phase the content of the synergid may already be in a state of degeneration. The second synergid may degenerate at the same time or has already degenerated before the pollen tube enters its companion. The pollen tube cytoplasm has already degenerated before the pollen tube enters the synergid. On entering the synergid, the pollen tube opens, through a pore or otherwise


(?), and the two generative cells are discharged. The vegetative nucleus degenerates if it has not done so earlier.

The reasons for synergid and vegetative nucleus degeneration are unknown. If autolysis is involved, what protects the male nuclei against it? One generative cell moves (how?) to the egg cell membrane and the other to the membrane of the central cell. The respective membranes fuse (again, how?), open up and the generative nuclei enter their respective receptor cells together with mitochondria, ER, ribosomes, vesicles, dictyosomes and plastids (Russel and Case, 1981, for Plumbago) which are contained in the cytoplasm of the generative cells.

We do not know if these organelles survive and what their function later on may be. It is only certain that in at least some cases, male plastids do survive. We also do not know if there is a difference in amount and species of male organelles and their survival between the egg cell on the one hand and the central cell on the other. Whereas the male nucleus entering the egg cell reaches the egg nucleus faster than its counterpart reaches the primary endosperm nucleus (or the two polar nuclei if these have not yet fused, in which case triple fusion will take place), fusion of egg and male nucleus is a slow process in comparison with the rapid fusion of the primary endosperm and its male nucleus. Is this difference really due to the more intense profusion of metabolic activity of the central cell as some authors (see Bhatnagar and Johri, 1972) believe?

Another question to be asked is: Is the role each male nucleus has to play preordained, or is it left to chance or accident which male nucleus goes where?

The many gaps in our knowledge regarding fertilization have also been noted by authors like Tilton (1981), who proposed a speculative model of the process which describes the sequence of events pertinent to our description as follows: Hormones produced by the ovary after pollination stimulate metabolic activity of the synergid(s) which produce lytic and chemotropic compounds. One synergid then autolyses. The filiform apparatus secretes both compounds into the micropyle to which the pollen tube is directed by the chemotropic compounds. The pollen tube's cytoplasm degenerates under the influence of the autolytic compounds and the pollen tube then enters the degenerated synergid through its filiform apparatus. The pollen tube opens, the male nuclei are released and migrate to their "respective male nuclei." Although this speculation is quite acceptable as such, it is filled with question marks which add to the many gaps in our knowledge which have been pointed out in the preceding pages. We may ask, for instance, what are the "hormones" involved? What triggers autolysis?

In summary, it must be stated that with the help of electron microscopy, intense modern investigation has brought to light many important details


of the fertilization process, but we still do not understand it because we know little or nothing of the physiological-biochemical processes involved, and of the controlling genetic and environmental factors. This is the more astonishing since fertilization is one of the most basic events in the plant's life.

Chalazogamy, which occurs in some important fruit trees like Pistacia vera, Juglans regia, etc., will not be dealt with because we know much less about the physiology of this type of fertilization than about that of micropylogamy.

Endosperm and Embryogenesis (Phase D of Fig. 1)

REFERENCE REVIEWS: Bewley and Black, 1978; Dure, 1975; Rubinstein et al., 1979; Wardlaw, 1955.

The development of endosperm and embryo has been described for many plant species (Bhatnagar and Johri, 1972; Johansen, 1950; Ma- heshwari, 1950; Raghavan, 1966; Wardlaw, 1955) thus there is no need to deal here with the descriptive aspects of this process. We will only indicate some problems concerning the physiological causality of the on- togeny of endosperm and embryo.

Developmental patterns

In his well reasoned work on embryogenesis, Wardlaw (1955) writes: "As a working hypothesis, a fertilized ovum may be regarded as a complex gene-determined reaction system. According to the components of the system and the sustaining environmental conditions, characteristic chains of reactions will be set in motion . . . . " Even today, over 25 years after it was written, this statement is fully acceptable, but it does not apply only to the fertilized egg. Embryos can develop from an unfertilized egg, from synergids, nucellar cells, integuments and there are even cases in which normal embryos develop side by side when one is derived from a fertilized diploid egg, the other from a haploid synergid (Cooper, 1943). Furthermore, cell cultures have shown that apparently any plant cell is able to give rise to embryos under the appropriate culture conditions. This shows that even differentiated plant cells are "omnipotent," containing all the genetic information needed to create whole new plants without fertilization.

The question remains therefore, what is the trigger mechanism causing the egg cell to develop an embryo? It cannot be exclusively fertilization as such, i.e. information transmitted to the egg nucleus by the sperm nucleus, although in the "normal" cases, fertilization seems to be the trigger. It must be something which is coupled to the fertilization process


but is present also independently; otherwise, there would be no cases of embryos developed from non-fertilized cells. Likewise, the trigger cannot be pollination since there are cases in which apomictic embryos develop without pollination. We certainly know much today about the hormones, enzymes, RNA, DNA transcription, translation, etc., involved in embryogenesis, but we still do not know what the specific "something" is which triggers embryogenesis.

There are many more unsolved problems concerning the start of embryo and endosperm genesis. One problem concerns the behavior of the zygote during the interval between its fertilization and first division. Zygote dormancy has already been mentioned. It may be of very different du- ration, i.e. from hours or days (seven days in Nicotiana) to several weeks as in Pistacia vera.

The reasons why some zygotes remain dormant and others divide more or less immediately after fertilization are unknown. In Gossypium and Nicotiana, zygote shrinkage was reported (Jensen, 1968a; Schulz and Jen- sen, 1968; Sehgal and Gifford, 1979). Jensen (1974) suggests that shrinkage in cotton is due to an initial osmotic disequilibrium between the egg cell and the developing endosperm. Wardlaw (1955) cites one most instructive case (Onagraceae), where the egg nucleus and the polar haploid nucleus are each fertilized by a generative male nucleus which supposedly contains identical genetic information; but the fertilized egg cell develops an embryo while the fertilized polar nucleus develops endosperm. Both must be pre-programmed for their respective developmental destiny--independently from the supposedly identical genetic information coded in the nuclei.

There are at least three possible explanations for this phenomenon. Either the generative cells bring in different plasmatic genetic information, or there is a topophysic effect involved, i.e. a difference exists in the "physiological conditions of the micropylar end and the center of the embryo sac" (Wardlaw, 1955); or there is an event which changes the "programme" of the one nucleus in relation to that of the other one during the development of the diploid archespore nucleus to the haploid nuclei of the embryo sac (transdetermination? metaplasy?). If topophysis is involved, the embryo sac, though small, would possess different mor- phogenetic "fields" which would impose their control upon the nuclei being positioned in a specific field. However, the question remains unsolved.

Another problem concerns the polarity of the egg cell. This finds its final expression in the fact that in most (or all?) angiosperm species the first step of embryogenesis is the formation of a transverse wall during the first division which divides the zygote into a most active apical cell, nearest to the chalaza, from which the many-celled embryo develops and


a basal cell, nearest to the micropyle, which by only a few divisions, develops into the suspensor "of unknown function" (Dure, 1975). The polarity is already evident in the fertilized egg cell before its first division: cytoplasmic metabolites and organelles active in protein synthesis (large polysomal complexes) accumulate in the apical pole, whereas the basal pole becomes vacuolated. We do not know the causal factor(s) of this polarization.

Jensen (1974) speculates that the appearance of the larger polysomes just mentioned is, in cotton, due to the release of RNA by the zygote nucleus in addition to the polysomes which persist during the first stages ofembryogenesis but disappear later. "These observations have intriguing possibilities in relation to information and differentiation" (Jensen, 1974). These most important "possibilities" have not yet been studied.

The apical and the basal cell created by the first division are very different. The apical cell contains many ribosomes, and mitochondria and plastids surround its nucleus, but has less ER than the basal cell which contains, in cotton, most of the tubular ER already formed before the first division of the zygote. The apical cell forms the embryo through a series of cell divisions, specific for each species, which proceed in such an "orderly manner" that it is almost possible to predict, at each stage ofembryogenesis of a given species, how and where the next division will take place. This enabled Sourges (1937) to establish his so called "laws of embryonomy." Apomictic embryogeny follows a similar "orderly" path, in contrast to seedling formation from single cell cultures ("artificial embryogenesis") where the embryo or seedling development is quite different from normal embryogenesis and proceeds in a non-orderly manner. Genetic factors alone, therefore, can hardly be the sole parameter re- sponsible for the orderliness in embryogenesis taking place inside the ovary. Could the cause be an embryogenetic "field" in combination with a "position effect that is where a given cell is in the embryo with relation to the surrounding cells?" (Jensen, 1974).

Another problem of embryogenesis concerns the so called "quiescent rudimentary embryos," i.e. the cases in which the embryo starts to develop but embryogenesis stops at a certain developmental stage and continues only after a period of dormancy. One could call this type of dormancy "exegetic embryo dormancy" (from the Greek, 'exegesis' meaning development) to differentiate it from "embryo dormancy," which relates to the dormancy of mature seeds. This exegetic dormancy is not identical with zygote dormancy. Exegetic dormancy of the embryo is not very rare. According to Martin (1946), it was found so far in 46 genera of angiosperms. The number of species in which the same phenomenon occurs must be much larger. In the genus Ilex, for example, 11 species showed it (Hu, 1975). In the case ofHeracleum sphondylium, the reason for this

SEED PHYSIOLOGY: OVULE TO M A T U R I N G SEED 161

dormancy is apparently energy starvation, i.e. nitrogen in the endosperm is in an insoluble form and becomes soluble and available to the embryo only after low temperature treatment (Stokes, 1952, 1953). In Ilex aquifo- lium, growth inhibitors "in the endosperm and/or in the membrane like testa" (Hu et al., 1979) interrupt embryogenesis.

It is not known how this inhibition is overcome. This is especially so in the case of Ilex opaca where excised embryos in the stage of exegetic dormancy continued their development in vitro in the dark and did not do so in light (Hu, 1976). This indicates that the Red-Far red mechanism is involved, but if so, why do the embryos become dormant in the ovary where they are supposedly "in the dark?" A good number of explanatory speculations could be made, but the facts which would support such speculations are missing. An interesting consequence of the exegetic embryo dormancy of Ilex is the fact that germination of the "mature" seeds is delayed and in some cases takes even eight years; the embryogenesis is only then being complete (Neal and Dye, 1964).

The recent paper by Hu et al. (1979) contains the highly interesting information that the ultrastructure of the exegetically dormant embryos " . . . closely resembles that of the quiescent mature embryos" of a number of other plant species of various genera, and that the ultrastructural changes occurring during growth reactivation resemble those typical for the initial germination phase of mature seeds. The authors conclude, therefore, that the ultrastructural patterns and their changes are typical for dormant embryos and embryos restarting growth, regardless of the developmental stage in which dormancy and renewed growth occur.

In nearly all cases the endosperm starts to develop before the first egg cell division. We do not know why this is so. We do know, however, that there is a tight metabolic link between mother plant (including integument and nucellus), and the endosperm and embryo development. This is true in those cases (e.g. Leguminosae) where the transient endosperm degenerates at a certain stage of embryogenesis and its final function, of storing reserve material for seedling growth, is taken over by the cotyledons and other organs of the embryo, and also for those cases where the endosperm persists (e.g. Gramineae). The importance of the system "mother plant- endosperm-embryo" for the nutrient flow into the developing seed will be discussed later. Some problems concerning the development of the endosperm remain.

The three types of the first phases of endosperm development (cellular, nuclear, helobial), typical for certain species, genera or families, differ in a most spectacular way. The physiological mechanisms involved in each case are completely unknown. This is also true for the causes of endosperm "digestion" in those cases where the endosperm degenerates at a certain stage of embryogenesis. If the endosperm is in these cases "digested by


the embryo," as is often stated, what is the triggering factor(s)? We may also ask whether it is the embryo which "digests" the endosperm in an active process, or whether the embryo "absorbs" the endosperm which degenerates and delivers its content to the embryo autonomously.

Metabolic processes

The storage organs of seeds and fruits are mainly the endosperm, the perisperm, the cotyledons, the embryonic axis and the radicle of the embryo. Since perisperm, which develops from the nucellus, appears only in a restricted number of species, it will not be dealt with in this paper.

The various substances in which energy is stored (carbohydrates, proteins, lipids) are sometimes evenly distributed in the storage organ and sometimes found in specific tissues. There is no case in which the energy reserves are exclusively of one kind, e.g. only starch.

Where the endosperm acts as the storage organ, its outer layer develops in some (all?) cases into a specific tissue, the aleurone layer, which, at least in the Gramineae, remains alive whereas in the mature stage of the achenes "some of the endosperm is dead" (Jacobsen et al., 1979). In the legumes, where the embryo is the final storage organ, the bulk of the endosperm is only a temporary nutritive tissue and disappears when the embryo, and specifically the cotyledons, become the sink for the various reserve materials. It is not clear if this is typical for all plants where the embryo is the food reservoir. Sometimes the endosperm is not composed of tissues as in the case of the coconut where it is a liquid syncytium.

It is quite astonishing to find, when checking the literature, that today's botanists, of all branches of the scientia amabilis, have, with the exception of the cases of cultivated plants, paid little attention to the morphology and anatomy of the mature endosperm, so that it is not even clear, for example, what part of the endosperm cells of the Gramineae is dead (see Jacobsen et al., 1979).

All the nutrients which accumulate in the storage organs and are partly transformed (polymerized) into energy-rich substances stored for later germination and seedling growth, are produced in the photosynthesizing apparatus of the mother plant and must be transported from there to the ovule and developing seed. The ovule stores some photosynthates before fertilization, and these are transferred to the embryo when parts of the maternal ovular tissues are "digested" (Thorne, 1980). The same is true for those cases in which the endosperm is a "transient reservoir" being digested during embryogenesis. But during embryogenesis most of the nutrients flow from the photosynthesizing organs straight to the embryo. This source-sink relationship is much more complex than what it seemed to be to the first investigators dealing with this question, and Dure (1975),


in his list of unanswered questions, rightly asks: "Are there unusual transport mechanisms involved in the flow of nutrients through the seed tissue to the embryo?"

This question is justified because there is no vascular connection between the vascular bundles and the embryo. The vascular bundles of the mother plant, through which the nutrients flow, end in the integument(s), the future seed coat. Thorne (1979, 1981) has studied the possible pathways of the photosynthates in soybean in which the whole seed coat is vascularized by a reticulate venation. This venation is composed only of phloem wherein the sieve tubes through plasmodesmata are symplastically interconnected with their companion cells. The phloem in the seed coat is surrounded by two distinct parenchyma tissues; "articulate parenchyma," which surrounds the phloem cells, and aerenchyma. The "articulate parenchyma" has many plasmodesmata. Its cells have a dense cytoplasm rich in mitochondria, dictyosomes, RER and vesicles, typical for cells "engaged in active carbohydrate transport and excretion" (Thorne, 1981). The aerenchyma contains large intercellular spaces which probably have a double function. In containing 02 they facilitate respiration, in containing CO2 produced by respiration of the embryo and the maternal tissues surrounding it, they may enable the cells of the aerenchyma to refix CO2. According to Hedley et al. (1975), seed coat tissues of peas contain PEP carboxylase needed for dark fixation of CO2.

Based on the structure of the seed coat tissue, Thorne (1980) proposes a model for the pathway of photosynthates. The photosynthates are "unloaded" from the sieve tubes into the companion cells, and from there into the articulate parenchyma. This process needs energy and is, as Thorne rightly states, "poorly understood." It is interesting in this regard that the four volumes of the new edition of the Encyclopedia of Plant Physiology dealing with "transport in plants" contain no special chapter on the transport into reproductive organs.

The transport through the aerenchyma into the symplast of the endothelium is apoplastic. This way of transport is, by its nature, slow (L~iuchli, 1976) and one may wonder whether it is efficient enough to satisfy the nutritional needs of the embryo. However this may be, the nutrient transport is certainly a limiting factor in the source-sink complex (Lichtner and Spanswick, 1981). From the endothelium the nutrients are transferred by diffusion to the cotyledons of the embryo via the multicellular tubules at the base of the embryo sac without passing through its protoplast.

The fact that no symplastic tie exists between the maternal and the newly developing sporophyte (Thorne, 1980) indicates, on the one hand, the independence of the new sporophyte from the controls of the maternal one which, on the other hand, controls the nutrient flow up to the point where it enters the embryonic cotyledons. Studies with excised developing


cotyledons (Lichtner and Spanswick, 1981) have shown that this last phase of nutrient flow, when sucrose is the transported photosynthate, is a sucrose/proton co-transport system where the driving force is a proton electro-chemical potential gradient.

The transport of photosynthates into the maize achene, where, in contrast to the soybean, the storage organ is the endosperm, has been studied by Felker and Shannon (1980). They concluded that sucrose, as in the soybean, is apparently the main transport-photosynthate. Sucrose moves from the end of the vascular bundle into thin-walled vacuolated cells of the pedicel (rachilla); from there, it continues through a "closing layer" consisting of "pedicel parenchyma and/or lower placento-chalazal cells" into placento-chalazal tissue to transfer cells (the "physiological bridge" between symplast and apoplast) of the basal endosperm- until finally reaching the endosperm. The pedicel cells have many plasmodesmata, as do the cells of the endosperm, but there is apparently no plasmodesmic connection between the maternal tissue and the endosperm. The placento- chalazal cells "appear" to be dead. The pedicel serves as a transient storage organ into which sucrose coming from the phloem flows symplastically, but Felker and Shannon (1980) do not exclude the possibility that apoplastic transfer is also involved. (My remark: If the placento-chalazal cells, which according to Felker and Shannon increase the free space, are dead, apoplastic transport through them seems probable). The transport from the pedicel to the symplast of the endosperm is certainly apoplastic. Some- where along the transport line sucrose is hydrolyzed into glucose and fructose, but it is not clear where the inversion takes place.

The unloading-uptaking of photosynthates in wheat differs apparently from that in maize, although both species belong to the same family. According to Jenner (1974a, 1974b) the photosynthates unloaded into the endosperm move there apoplastically, and sucrose hydrolysis occurs at a later stage than in maize. These facts underline the difficulty of deter- mining to what extent results--obtained with very few species--can be generalized.

Many factors can be limiting to the transport ofphotosynthates on their way from their place of origin to the storage organs of the maturing diaspore (Hardham, 1976; Liu and Shannon, 1981; Streeter and Jeffers, 1979). The slow rate of apoplastic transfer has already been mentioned as one such potentially limiting factor. But there are many more. Pho- tosynthesis and translocation of the photosynthates in the phloem before their arrival at the place of unloading are processes which are exposed to the impact of the environment and environmental parameters, and can therefore easily become limiting factors affecting the loading of the storage organs. This indirect environmental control of the specific processes of nutrient deposition and accumulation in the storage organs has been large-


ly neglected although our knowledge of the impact of the environment on photosynthesis and transport in general, is vast. Only a few authors-- like Jenner (1974a, 1974b) for wheat, and Thorne (1980) for soybeans-- have pointed out that "photosynthate availability may at times limit seed growth and productivity" (Thorne, 1980). There are no quantitative data about this limitation although it is known in some cases how much photosynthate produced by the leaves is transported to the developing seeds. In soybean, for example, this amounts to 7-20 mg sucrose seed-~ day-~, which is about half the sucrose production of 1 dm 2 leaf day-~ (Egli, 1975; Thorne, 1980).

It is not yet clear to what extent endogenous hormones, especially cytokinins, are involved in nutrient transport to developing seeds, although it is known that they play a role in the mobilization of nutrients. Many seeds contain cytokinins which probably are necessary for embryo growth (see literature in Crosby et al., 1981). In soybeans, flower and fruit abscission are very common (Abernathy et al., 1977; Adedipe et al., 1976; Wiebold et al., 1981) and could be prevented by application of exogenous cytokinin (Crosby et al., 1981). Adedipe et al. (1976) showed that in cowpeas the application of abscission-preventing cytokinin stimulated sucrose transport in a cultivar with a high percentage of abscissing fruits, but had no effect on sucrose transport in a low abscissing cultivar. This suggests that, for soybeans and cowpeas, the application of exogenous cytokinin supplements the endogenous cytokinin "increasing the ability to competitively mobilize nutrients" (Crosby et al., 1981). If so, the function of the endogenous cytokinin would be to control the competition for nutrients between growing fruits and growing vegetative organs.

Besides photosynthesis, all the other elements needed for the development of the seed and their storage materials are transported from the mother plant to the maturing seeds. Details of this process and the relation between source and sink are most meager. Nitrogen, because of its importance for protein synthesis, is given here as an example. Hay et al. (1953) have already stated that in maize, at pollination time, the mother plant has already accumulated enough N to satisfy the needs of the achenes. But in spite of the fact that there seems to be a relationship between leaf nitrate reductase and leafproteinase activity (Reed et al., 1980), the details of this process are not known. It is also not known how the environment and the developmental phases of mother plant and achenes affect N-transport and accumulation.

In summarizing this section on nutrient transport, the following questions concerning the main gaps in our knowledge remain to be asked:

1) What are the mechanisms of transport from symplast to apoplast to symplast? The same question has already been asked by Dure (1975) and still remains unanswered. But not only the transport symplast-apoplast-


symplast remains a problem. Even the apoplastic and symplastic transports by themselves are still not well understood although we know that complex membrane mechanisms are involved. In reviewing apoplastic and symplastic transports in tissues, L/iuchly (1976) writes: " . . . critical assessment of the role of the apoplastic p a t h w a y . . , must base itself on cell wall chemistry and cell wall cytology incorporating biophysical con- s idera t ions . . , it may not be too surprising that such a treatment brings up more questions than solutions." And: "It may reasonably be concluded that the principle of symplastic transport is well established. However, the information required for a quantitative treatment of the system is lacking" (Spanswick, 1976).

2) Does the fact that the photosynthates and nutrient elements flow from a 2n mother plant to a 3n tissue to a 2n (~ + ~) embryo indicate that each phase of the transport process is under the exclusive control of the respective genomes controlling membrane structure and function or is there a kind of co-ordinated action?

3) Is it really true that "the proliferation of the nucellus and/or endosperm results from the nutrient flow from the vegetative plant" (Dure, 1975), or is it the other way around? What is cause and what is effect? What is the triggering mechanism and what sets it in motion?

4) How does the environment directly or indirectly affect the nutrient flow?

Concluding Remarks

This review has tried to summarize what modern botany knows about the developmental physiology from ovule to maturing seed and to point out the way to future research by laying bare unsolved problems of which there are many.

Besides the many unanswered questions contained in these pages, there remains the question which any developmental physiologist has asked himself at one time or another, perceiving the "ordered complexity" (Wardlaw, 1955) and ordered sequence of events, i.e. the "regulated rep- lication, transcription and translation" (Shatkin, 1981), so typical for all developmental processes: Are there supergenes or any other entities (in- trons, mini-chromosomes, cap structures? see Shatkin, 1981) which, in a preordained way, determine at what moment during the chain of developmental events a given gene has to be triggered to express itself phenotypically, a process which by itself is of a most complex nature?

When reviewing the history of our knowledge on seed physiology from antiquity to modern times, one gets the impression that the progress in this field (Evenari, 1980/81) happened in quantum jumps. During the Greek-Roman period, when one of the foundations of Western civilization


was laid, the first observations of seed physiology were made. The mo- tivation for this was partly the keen interest of the Greeks in observing nature and their attempt to understand its underlying "reason," and partly the need to solve practical problems of agriculture.

After a long interval, the next jump took place when humanism was born and with it, the urge to understand nature in a rational way. As far as seed physiology is concerned, this was only possible because that period coincided with the development of physics and chemistry and with the invention of the light microscope, giving physiologists the technical means to observe, for the first time, the inner structure of organisms and to probe their working mechanisms by experiment.

The next jump occurred with the invention of the electron microscope [The electron microscope was first used in 1934; apparently the first to use it on seeds was Cavazza (1950) followed by Stein and J ancke (1958).], which enabled the biologist to penetrate to the level of submicroscopic organization and with sophisticated physico-chemical technology (ultra- centrifuge, electrophoresis, immuno-chemistry etc.) helped create molec- ular biology. This review deals with the impact that this quantum jump had on our knowledge of the development from ovule to maturing seed.

In reviewing the great progress made in our time on the whole biological front of research, including the small part of it which has been reviewed in this paper, the reviewer asks himself how it is that with each step forward into the yet unknown phenomena which seemed to be simple turn out to be more complex, and solutions found to any one problem pose new questions. Du Bois-Reymond, a great physiologist of the past century (1818-1896) has illustrated this in a fitting simile: A man faces a closed door and tries relentlessly to open it. He succeeds--and finds himself in a passage-way leading to other closed doors.

And so scientists, driven by their curiosity, will continue their never- ending quest for knowledge.

Acknowledgments

My thanks are due to Prof. Dr. A. Meyer of the Botany Department of the Hebrew University for his critical review of my manuscript.

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Seed physiology: From ovule to maturing seed