The heterocysts of blue-green algae (Myxophyceae).

Biol. Rev. (1975),50, p p . 247-248 BRC PAH 50-7

THE HETEROCYSTS OF BLUE-GREEN ALGAE (MYXOPHYCEAE) BY V. V. S. TYAGI Department of Botany, Government College, K o t a (Rajasthan), India (Received 7 August I 974) CONTENTS

. . . . . . . . . 11. Structure . . . . . . . . . . (i) Cytological aspects . . . . . . . . (ii) Comparison with akinetes and vegetative cells . . . 111. Biochemical composition . . . . . . . . (i) The wall . . . . . . . . . . (ii) Pigments . . . . . . . . . . (iii) Lipids, proteins and nucleic acids . . . . . . IV. Differentiation . . . . . . . . Control mechanisms . . . . . . . . . (a)Nutritional control and determination of pattern ( b ) Photoregulation . . . . . . . . (c) Metabolic control . . . . . . . . V. Functions. . . . . . . . . . . (i) Archaic reproductory structures . . . . . . (ii) Storage cell . . . . . . . . . (iii)Attachment organ . . . . . . . . (iv) Regulation of akinete formation (sporulation) . . . (v) Nitrogen fixation . . . . . . . . VI. Geological history . . . . . . . . . VII. Conclusion . . . . . . . . . . VIII. Summary. . . . . . . * . . . IX. References . . . . . . . . . . I. Introduction

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INTRODUCTION

The Myxophyceae or Cyanophyceae (commonly called the blue-green algae) are prokaryotic plants the structure and metabolism of which have been the subjects of extensive investigation in recent years. Of the many problems presented by this group, that afforded by the heterocysts has attracted considerable attention. These distinctive cells are found in the filaments of many species of blue-green algae and are distinguishable from vegetative cells mainly by their larger size, thicker wall, and more homogeneous cell contents. Thuret (18++)wasprobablythe first to record the presence ofheterocysts in algal filaments. Fritsch (1904b) found that the first heterocyst differentiates from a terminal cell of the spore germling. The vegetative cells progressively divide, contributing to the length of the filament, and the second heterocyst is formed from a cell situated at a specific distance (measured in terms of number of vegetative cells) 16

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away from the first. Consequently, heterocysts are distributed along the length of the filament with characteristic cellular spacings between them. The taxonomic value of the presence or absence of heterocysts was recognized a long time ago and a classification of the order Hormogonales into sub-orders Homocysteae and Heterocysteae was suggested (Bornet & Flahault, 1886). Although this classification was later abandoned, since it separated many taxa which have otherwise good affinities, the heterocyst remains a distinctive feature in identifying some genera and species. T h e importance of these cells in myxophycean growth and development was disregarded until about thirty years ago, although some suggestions concerning their role had been made (Fritsch, 19oqb; Troitzkaia, 1924).A beginning to the understanding of their biological role was made by Fogg (1942, 1944,1949)when he indicated that these cells are specifically produced in response to changing growth conditions. Sufficient uncertainty followed, however, to impel Fritsch (1951)to call the heterocyst a ‘botanical enigma’. Since then, the situation has been changing; the fine structure of the heterocyst has been revealed and much information now exists on its metabolic aspects. Our knowledge of heterocyst differentiation is still meagre, but biologists are becoming aware that these cells can provide a model system for studying developmental processes under simple experimental conditions (see Singh, 1974). Interest in the study of the heterocyst has been enhanced recently by observations on the functional linkage between heterocyst formation and nitrogen fixation (see Stewart, 1972, 1973; Fay, 1973 for review). T h e literature on the ultrastructure of the heterocyst and on its metabolic aspects has been periodically reviewed (Lang, 1968; Fay, Stewart, Walsby & Fogg, 1968; Stewart, 1972). Fogg, Stewart, Fay & Walsby (1973) and Fay (1973) have dealt with various aspects of the biology of the heterocyst. Cytological aspects of heterocyst formation were critically reviewed by Lang (1972), and a discussion on their taxonomic value in Myxophyceae is contained in a paper by Whitton (1969). It is the aim of this review to examine the present state of our knowledge of the structure, differentiation and function of the heterocyst. 11. STRUCTURE

T h e heterocyst is generally slightly larger than the vegetative cells, I n shape, the heterocysts generally resemble the vegetative cells from which they have originated. I n such genera as Anabaena and Nostoc and also in the Rivulariaceae the heterocysts are almost round, whereas in others, e.g. Aulosira, Scytonema and Hapalosiphon they are more rectangular. T h e heterocysts of several species are mostly enclosed in the thick sheath of the filament. I n genera in which thick-walled resting spores (akinetes) are formed, the heterocysts are found generally in close proximity to the akinete. I n some genera as a regular feature (e.g. Anabaenopsis arnoldii Aptekarj) and in others as an abnormality, the heterocysts occur in pairs and occasionally in chains. (i) Cytological aspects Structurally, the heterocyst appears simple in the light microscope, though finestructural studies have shown this to be illusory. T h e contents of a mature heterocyst

The heterocysts of blue-green algae (Myxophyceae)

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Fig. I.Diagram of a portion of a longitudinal section of a heterocyst showing various layers of the wall and lameltar organization at the polar end. Lz-Li,, layers of the inner wall;f, fibrous, h, homogeneous, and Z, laminated layer of outer wall (terminology of Lang & Fay, 1971).pl, plasmalemma; pd, microplasmodesmata; pp, polar plug; ct, contorted thylakoids ; t, normal thylakoids. Based on an electron micrograph by Wilcox et al. (19736).

are homogeneous and pale yellow. Its wall is distinctively thicker than that of the vegetative cells and conventionally two layers are recognized in it. Muhldorf (1937) and Fritsch (1945) believed the outer layer to be the original one belonging to the vegetative cell (since heterocysts originate from vegetative cells) while the inner layer was gradually secreted toward the outer wall by the membrane of the cell during heterocyst development. Recent studies of fine structure have, however, revealed that when a vegetative cell differentiaties into a heterocyst, its wall remains intact and the second wall (also called envelope) is formed by deposition of material externally to the pre-existing wall (Lang, 1965). Lang & Fay (1971) have shown that the outer wall of the heterocyst of Anabaena cylindrica Lemm. is composed of three distinct layers: a peripheral fibrous region, an underlying thick homogeneous layer, and a laminated layer which borders on the inner wall (Fig. I). The appearance of the three layers also takes place in that sequence during heterocyst development (Kulasooriya, Lang & Fay, 1972; Wilcox, Mitchison & Smith, 1973 b). The inner wall resembles in ultrastructure the wall of the vegetative cell in that, like the latter, it is also composed of four layers (Li-Liv). Inner to the wall layers and surrounding the protoplast of the heterocyst is the plasmalemma. All the layers of the outer wall surround the heterocyst completely except at the junctions of the heterocyst with the vegetative cell (Winkenbach, WoIk & Jost, 1972). 16-2

V. V. S. TYAGI T h e heterocyst possesses one or two pores in communication with adjacent cells. The intercalary heterocysts have two pores, one at each pole, while the terminal ones possess only a single pore toward the filament (as in Riuularia). Some abnormal intercalary heterocysts with three pores have been observed in Brachytrichia balani (Lloyd) Born. & Flah. and Mastigocladus laminosus Cohn. (both of the Mastigocladaceae) (Iyengar & Desikachary, 1953;Venkatraman, 1957);each pore in these cases connects with a vegetative branch. Formation of three-pored heterocysts has also been induced in Nostoc linckia (Roth) Born. & Flah. by ultraviolet irradiation (Singh & Tiwari, 1969). The significance of such abnormalities is not yet understood, and detailed studies on their occurrence and distribution in different genera and on any environmental factors leading to their formation might be informative. Usually the terminal heterocyst possesses a single pore, but when the intercalary heterocyst becomes terminal, due to fragmentation of the filament at that point, obliteration of the pore on the free end has been observed (e.g. in Anabaenopsis arnoldii var. indica, Ramnathan, 1938). Muhldorf (1937),while discussing the nature of heterocyst pores, considered them as ‘true ’ connexions between the heterocyst and the vegetative cell. A real cytoplasmic continuity was also visualized by Fritsch (1951)between heterocyst and vegetative cell through the pore. T h e most detailed studies with the light microscope on this aspect were those of Fogg (1951);he showed histochemically that the pore channel is lined on the inside by the inner wall, but found no cytoplasmic continuity. Micrographs of thin sections show that the pore channel is surrounded by all the layers of the outer and inner walls, but at the junction with the vegetative cell only L, and L,, of the inner wall and the plasmalemma are present (Lang & Fay, 1971).The possible existence of plasmodesmata connecting the plasmalemmas of the heterocyst and the adjacent cells, first envisaged by Geitler (1938),has recently been confirmed by electron microscopy (Wildon & Mercer, 1963~; Lang & Fay, 1971).These seem to have a role in exchange of physiologically important substances between the two cell types (Wolk, 1968). The homogeneous and laminated layers of the wall are thickened around the pores, and in electron micrographs the pore channel is seen to be almost ‘plugged’ with electron-transparent material (in unstained sections). T h e ‘ plug materials ’ appear as shiny granules and are generally referred to as polar granules. T h e granules are highly refractive like the cyanophycin granules, and for this reason some earlier workers had considered them to be cyanophycin granules of large size and high refractive index. However, Baumgiirtel (1920)and Geitler (1921a) regarded them as special structures composed of an outer layer of nucleoglycoproteinaceous matter and the inner one of protein only. Fritsch (1951) attributed the appearance of the granules to the accumulation of the building material of the cell wall. Fogg (1951) showed that the polar granules abound in material which reacts positively to stains for arginine and to the Feulgen reaction, but unlike cyanophycin granules these are insoluble in acids (see, however, Wolk, 1973).I n recent years few details of the composition of the granules have been worked out, but in the light of the physiological relationship between the heterocyst and adjacent cells it is probable that these bodies have special permeability properties. The structural changes during heterocyst differentiation in Anabaena cylindrica were explored by Fogg (1951)using cytological staining and ultraviolet photomicro-

The heterocysts of blue-green algae (fWyxophyceae)

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graphy. Formation of the heterocyst was found to begin with the enlargement of the vegetative cell accompanied by migration of cellular material towards the poles which gives it a homogeneous appearance. In the intermediate stages, this material concentrates near the junction between the developing heterocyst and the adjacent cell, and in the end possiblymoves into the latter. Earlyworkers had also observed a homogeneity in the heterocyst protoplasm on staining with various stains (e.g. Feulgen stain ;Poljansky & Petruschewsky, 1929); this led to the belief that heterocysts contain quantitatively less chromatin than other cells. Kumar (1962a) has investigated heterocyst differentiation in Camptylonema lahorense Ghose with the light microscope and found an increase in cell size, disappearance of granules, and the biosynthesis of an additional wall as the main stages. The formation of the pore channels and organization of polar granules are the phases last to be completed. In this alga, division of a mature heterocyst was also observed (Kumar, 1962b). Degradation of polyphosphate granules was observed during differentiation (Talpasayi & Bahal, 1967), but its mechanism and the fate of the products remain to be elucidated. Grilli (1964) and Lang (1965) have described the ultrastructural changes during heterocyst differentiation in Anabaena azollae Strasburger. These workers agreed that cell enlargement, gradual disappearance of granules (Polyhedral bodies, polyphosphate and cyanophycin granules), reorientation of the thylakoids into a honeycomb configuration at the polar ends and synthesis of outer wall-layers are the main events accompanying heterocyst differentiation. More recently, Kulasooriya et al. (1972) and Wilcox et al. (19736 ) have studied the ultrastructural changes in the developing heterocysts of Anabaena. Wilcox et al. (1973b) have defined 7 stages in development in terms of certain distinctive ultrastructural features. I n stage I, the laying down of an outer fibrous layer occurs around an otherwise normal vegetative cell. This is completed by stage I1 when the junction between the developing heterocyst and the vegetative cell is ‘squared off’. In stages I11 and IV, the junction is drawn out in the form of a neck, a homogeneous layer is synthesized below the fibrous, and the lamellae containing electron-transparent intralamellar spaces appear throughout the protoplast. The laminated layer of the wall is synthesized in stage V and there also occurs a concentration of contorted membranes in the polar regions. From these regions a proliferation of new lamellae may be seen in the maturing heterocyst. Heterocysts in stage VI show deposition of an electron-transparent material in the pore channel and also the formation of microplasmodesmata. I n the final stage the heterocysts reach maturity with some proliferation of lamellae and completion of the organization of material in the pore channel. The account of developmental stages given above probably applies to the heterocysts of most genera. I t seems to deviate, however, from that in Chlorogloea fritschii Mitra, a genus of special interest since it was once believed to lack heterocysts and so was placed in the Chroococcales (see Fritsch, 194s). After the discovery of heterocystous stages (Fay, Kumar & Fogg, 1964), a study of heterocyst development in this alga was undertaken by Whitton & Peat (1967). Two kinds of heterocysts were distinguished: one in the filamentous form of the alga, termed H - I , and the other in the dark-grown endospore stage, termed H-3. The two kinds exhibited different

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internal structure in that the heterocysts of the endospore stage possessed a higher density of granular inclusions and also a dense thylakoid system (characteristic of vegetative cells). I n fact, the heterocysts of the endospore stage are incompletely differentiatied and represent merely a stage in development. Further these studies indicate that the characteristic developmental sequence may be arrested at certain stages, and that the heterocyst may undergo differentiation to a limited extent in the dark (see p. 264). T h e heterocysts of Nostoc muscorum Ag. ex Born. & Flah. have also been shown to exhibit a similar pattern of development in the dark (Ginsberg & Lazaroff, 1973). I n this case, the cytoplasmic contents of heterocysts in 58-day old darkgrown filaments were found similar to those of the vegetative cells except that they appeared to lack cyanophycin granules and polyhedral bodies. On transfer to light, loss of granules and further differentiation takes place. The structural integrity of the heterocyst may be damaged to different extents according to the method employed in its isolation for electron microscopy (Fay & Lang, 1971). The commonly used mechanical methods (viz. French press, sonication) and osmotic shock cause disorganization of the thylakoid system. Isolation by means of lysozyme (a hydrolytic enzyme which acts on the walls of the vegetative cell but not on that of the heterocyst) has produced heterocysts of intact internal structure. The structural details of the wall of the heterocysts of Anabaena cylindrica given earlier were studied in material isolated by this method (Lang & Fay, 1971). Lang (1972) has also observed gas vesicles in immature heterocysts of Anabaena flos-aquae (Lyngb.) Bren ; their numbers, however, gradually declined with maturation. Lipid droplets, polyglucan granules and phycobilisomes have been recorded from immature heterocysts but are absent from those that are fully developed. Ribosomes, on the other hand, seem to be present in all stages of heterocyst development (Lang & Fay, 1971). (ii) Comparison with akinetes and vegetative cells During the differentiation of an akinete of Cylindrosperrnum (Miller & Lang, 1968; Clark & Jensen, 1969) and of Anabaena sp. (Lang, 1972) layers of a thick fibrous envelope are deposited externally to the cell wall, the thylakoids are mostly retained, and cyanophycin granules enlarge. Comparative studies on the infrastructure have shown that whereas the heterocyst possesses few granules and some membranes, the akinetes have large granules (polyhedral bodies and polyglucan granules) occupying most areas of the cell (Wildon & Mercer, 1963~).The massive outer envelope of the akinete is composed of original sheath material and a new fibrillar component. Unlike heterocysts the akinetes lack pores and polar granules. Structural differences between the heterocyst and the vegetative cell form the basis of our definition of the heterocyst. I n the vegetative cell the thylakoids form an anastomosing network and there are several kinds of granules, e.g. cyanophycin, polyglucan and polyphosphate in the lamellar system, whereas these are absent from the heterocyst (Wildon & Mercer, 1963b; Lang, 1968). Further, the heterocyst lacks the profuse thylakoid system characteristic of the vegetative cell and has no gas vacuoles. There is no definite evidence on the organization of nuclear material in the heterocyst, although aggregates of DNA fibrils are known in the nucleoplasmic region of the vegetative


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cell (Leak, 1965; Fuhs, 1969). Further differences between the two cell types lie in their susceptibility to various lytic agents. As already mentioned, the walls of the vegetative cell are destroyed by the hydrolytic enzyme lysozyme, whereas the heterocyst remains unaffected (Jensen & Sicko, 1971, Fay & Lang, 1971). Similarly, the viruses specific to the blue-green algae and certain myxobacteria, digest the vegetative cell wall but leave the heterocyst intact (Singh & Singh, 1967; Daft & Stewart, 1971, 1973). Most of these pathogens also fail to lyse the akinetes. 111. BIOCHEMICAL COMPOSITION

The heterocyst is remarkably different in its biochemical composition from the vegetative cells and the akinetes. Early workers employed classical staining methods for the localization of structural ingredients in the heterocyst (Geitler, 1921b ; Bharadwaja, 1933). With the improved techniques of biochemical analysis, some new chemical derivatives, hitherto unknown from plant cells, have been discovered in the heterocyst. (i) The wall According to Dunn & Wolk (1970) the wall is estimated to account for about 52% of the dry weight of the heterocyst. These workers have further found that the isolated envelopes of Anabaena cylindrica have ca. 62% carbohydrate, 4% amino compounds, I 5 % lipid, and 2 % ash. Corresponding values for the vegetative cell are approximately 18, 65, 3 and 2.5%. The monosaccharide composition of the polysaccharides from the heterocyst wall is: glucose 73 yo,mannose 21 %, galactose 5 yo, xylose 8% and fucose 2%. This is significantly different from the corresponding composition of the vegetative cell wall, but similar to that of the spore. On this basis, the authors have concluded that the additional layers of the heterocyst wall are composed chiefly of a glucose-rich polysaccharide. The polysaccharide seems to be different from cellulose since the envelope-wall fraction of the heterocyst is neither dissolved by 85 % phosphoric acid nor is digested by the enzyme cellulase (Fogget al., 1973), although at one time it was considered to be mainly cellulose (see Fritsch, 1945 for references). The lipids of the heterocyst wall are localized chiefly in the laminated layer (Winkenbach et al., 1972; Lambein & Wolk, 1973). The inner wall can account for the amino compounds and some carbohydrates ; the large amount of carbohydrate has therefore been attributed to the fibrous and homogeneous layers (see Wolk, 1973). The decrease in the percentage of amino compounds in the heterocyst wall is also significant. Since the wall of the vegetative cell contains chiefly amino compounds it will be interesting to investigate whether this wall also undergoes biochemical change during differentiation. In the analysis of Dunn & Wolk (1970) the walls of the heterocyst were found to have significantly more polysaccharides than those of the akinetes, but the monosaccharide composition of both cell types was strikingly similar. This suggests a polysaccharide component common to the walls of the heterocysts and akinetes. On the other hand, akinetes contain six times as much amino compounds as the heterocysts. The carbohydrate and amino fractions together account for about 65% of the wall material of both cell types.

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There is no definite evidence on the composition of the inner wall-layers of the heterocyst, although ultrastructurally they resemble those of the vegetative cell (Lang, 1965). Dunn, Simon & Wolk (1971) have, however, indicated that during heterocyst formation the cell wall and especially its peptidoglycan-containing L,, is largely retained. (ii) Pigments Fritsch (1951) believed that the pallor of the heterocyst is due to the disappearance of pigments other than the carotenoids, during its formation. Recent studies have shown that p-carotene is abundant, myxoxanthophyll is absent, whereas phycocyanin assumes a negligible proportion in the heterocysts of Anabaena cylindrica (Fay, 1969). I n this alga, the heterocysts still contain about 77% as much chlorophyll as do the vegetative cells but lack phycocyanin (Wolk & Simon, 1969). Microspectrophotometric studies in vivo on the pigment composition of Anabaena sp. L-31 suggest that the heterocysts lack allophycocyanin (A,, = 650 nm) and the short-wave form of chlorophyll a (A,,, = 666 nm) which are pigments invariably associated with vegetative cells (Thomas, 1970, 1972). However, C-phycocyanin (A,, = 620 nm) is present in small quantity under certain conditions (see Thomas, 1972); it may indeed be present under all conditions but sometimes bleached by endogenous reductant = 580 nm) is almost (Winkenbach et al., 1972). Similarly, phycoerythrin (A,, completely absent. The amount of carotenoids (460-490 nm), however, remains almost unaltered (Thomas, David & Gopal-Ayengar, 1972). Obviously, the pigments that disappear during heterocyst formation are those associated with the activity of photosystem I1 in the blue-green algae. Donze, Haveman & Schiereck (1972) have found relatively more of the P,,, form of chlorophyll a in the heterocyst, the pigment associated with activity of photosystem I. These findings together with those of Scott & Fay (1972) provide evidence that the heterocysts ‘photosynthesize’ with only one functional photosystem. (iii) Lipids, proteins and nucleic acids Some heterocystous blue-green algae have been found to possess non-saponifiable glycolipids not hitherto known in non-heterocystous genera or in the foliage of higher plants (Nichols &Wood, 1968). These observations engendered the idea of a relationship between these glycolipids and the heterocysts, since the latter formed the main distinguishing character of the algae possessing them. Wolk & Simon (1969) found two previously unknown lipids in significant amounts in the heterocysts of Anabaena cylindrica. Further examination of some heterocystous genera revealed that the glycolipids, along with a small quantity of an acyl lipid, form their principal lipophilic component (Walsby & Nichols, 1969). Moreover, four classes of lipids (mono- and digalactosyl diglycerides, phosphatidyl glycerol, and a sulpholipid - all containing a-linolenic acid), which are the main lipid fractions in the vegetative cells and are found in the chloroplasts, are absent from the heterocyst. However, they are also absent from the photosynthetic bacteria (Kenyon & Stanier, 1970). T h e heterocystspecific glycolipids have been found to be mono-glucoside and galactoside derivatives of long-chain polyhydroxy alcohols. T h e major alcohol moiety in these lipids is


25 5

I ,3,25-trihydroxyhexacosanewhich is accompanied by much smaller quantities of I ,3,25,27-tetrahydroxyoctacosane- these polyhydric alcohols seem never to have been

detected in plant cells (Bryce, Welti, Walsby & Nichols, 1972). The exact role of the heterocystous lipids in metabolism is uncertain, although it is possible that they are involved as carriers and intermediates in cell-wall biosynthesis - a function analogous to that of bactoprenol in bacteria (Bryce et al., 1972). Much in accordance with such a proposition, the heterocyst-specific glycolipids have been localized in the laminated layer of the envelope (Winkenbach et al., 1972;Lambein & Wolk, 1973). The chemical nature of the acyl component remains unknown. T h e possibility that the lipids of the heterocyst are associated with the lamellae and have a role in maintaining the anaerobic environment in the cell has also been envisaged (see Lang, 1972). No chemical analysis of the heterocyst has been made specifically with regard to its protein composition. Much of our knowledge on this aspect is indirect, being derived from studies with specific stains or from the metabolic (enzymic) activity of the heterocysts. Fogg (1951),using a modified Sakaguchi test, demonstrated the existence within the heterocyst of some arginine-containing substances. T h e nature of these substances was, however, not clear at that time. It is now evident that the arginine-containing substances are proteins constituting the cyanophycin granules, and are redistributed throughout the cytoplasm after disorganization of the granules during heterocyst development (Simon, 1971).Histochemical techniques have also been used to disclose enzyme activity in the heterocysts. Drawert & Tischer (1956) observed that heterocysts reduce triphenyl tetrazolium chloride (TTC) and oxidize the Nadi reagent faster than the vegetative cells, and suggested the presence of active enzymes in the heterocyst. Tetrazolium reduction has been recently confirmed in the heterocysts of Anabaena cylindrica, A. inequalis (Kutz) Born. & Flah., A . variabilis (Kutz) Born. & Flah. and Anabaenopsis circularis (West) (Fay & Kulasooriya, 1972). Heterocysts respire actively and also reduce acetylene to ethylene - activities only possible with organized enzyme systems (Fay & Walsby, 1966;Stewart, Haystead & Pearson, 1969). T h e information on the presence of nucleic acids in the heterocysts is very meagre. T h e developing heterocyst gives a positive reaction to the Feulgen and haematoxylin reagents and this becomes insignificant in the fully mature heterocysts (Fogg, 1951). The electron micrographs do not depict any nuclear material as conspicuous as that in the vegetative cells (Lang, 1965), nor is histochemical localization much more informative. Leak (1965)has however shown, using autoradiography, the incorporation of tritiated thymidine in the nucleoplasmic region of the heterocyst. Much more information is required on nucleic acid metabolism in the heterocyst for a clear understanding of its differentiation and function. IV. DIFFERENTIATION

T h e heterocysts originate from vegetative cells during the growth of cultures and are regarded as a case of adaptation with a persistent phenotypic expression. T h e formation of heterocysts is a special case of differentiation, since here the cytoarchitectural changes are confined to a single cell. Certain aspects of heterocyst differentiation

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are known in some detail, but these are too meagre to warrant any definitive conclusion about the control mechanism of the process. Some nutrients affect their differentiation, but in the absence of proper knowledge of the endogenous control system nothing can be said with confidence about the role that they play. Nevertheless, the geometrical simplicity of the heterocyst and the ingenious methods of experimentation can make this subject a fascinating study.

Control mechanisms With the exception of the Rivulariaceae and some other genera where the only heterocyst is basal (or terminal), most other families possess intercalary heterocysts. In these families heterocyst formation is a sequential process in growing cultures; new heterocysts differentiate after the attainment of a specific quota of vegetative growth (Fogg, 1944).Under a set of environmental conditions the cellular distance between two heterocysts remains constant and heterocyst formation occurs about half-way between the two adjacent ones. The frequency of the heterocysts in filaments is generally expressed as the number of heterocysts per hundred vegetative cells. This frequency readily changes with the various environmental parameters that affect growth and is therefore a sensitive measure of the capacity for heterocyst production by cultures under any given set of experimental conditions (Fogg, 1949; Tyagi, 1 9 7 3 ~ ) . Fogg (1944) concluded on the basis of studies on Anabaena cylindrica that the determining force for heterocyst formation resides in cell division (in the filament). Any agent of the cell’s environment which affects cell division also influences heterocyst production. Fogg (1949)further believed that heterocyst frequency depends on (i) rate of growth in cell numbers and (ii) rate of heterocyst differentiation. Extrinsic stimuli which interfere with growth lead to significant variations in heterocyst frequency but some factors may influence the frequency by affecting the process of differentiation alone. ( a ) Nutritional control and determination of pattern Canabaeus (1929) investigated the effects of some mineral salts on the heterocysts of blue-green algae. Chlorides of sodium and potassium caused an increase in heterocyst size and frequency up to a certain concentration (0.2%), beyond which the effects reversed. Similarly, anaerobic conditions in the dark led to an increase in the size of the heterocysts. I n Anabaena doliolum Bharadwaja a gradual and limited increase in heterocyst size has been observed in increasing concentrations of molybdenum (Tyagi, 1 9 7 4 ~ ~These ). findings, though interesting, do not provide any significant clue to the mode of heterocyst differentiation. The nature of the environmental factors controlling heterocyst formation has been more thoroughly investigated by Fogg (1942, 1944, 1949). He found that substances providing a readily assimilable source of ,nitrogen inhibited heterocyst production, and an inverse relationship existed between heterocyst frequency and the concentration of nitrogen source in the medium. Nitrate, glycine and asparagine caused only a transient inhibition reflected in a slight decrease of heterocyst frequency, whereas the


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inhibition by ammonium chloride was more permanent. T h e latter lasts as long as an appreciable concentration of the substance remains in the medium. Glucose and succinic acid which provide assimilable carbon, on the other hand, produced a transient increase in heterocyst frequency when added to the medium. Fogg (1949), therefore, formulated the hypothesis that ammonia is the main inhibitor and that its endogenous level controls heterocyst formation. T h e stimulatory effect of carbon sources on heterocyst production was attributed to a temporary preponderance of intermediates of carbon metabolism to which the ammonium nitrogen may be linked. The observations of Fogg have since been confirmed by several workers using other heterocystous algae. I n Camptylonema lahmense and Anabaena jos-aquae critical levels of combined nitrogen were found to block heterocyst formation (Pandey & Mitra, 1962; Mickelson, Davis & Tischer, 1967). The role of ammonia as the most powerful inhibitor was further confirmed in Anabaena ambigua Rao (Talpasayi & Kale, 1967) and Clzlorogloea fritschii (Peat & Whitton, 1967). Singh and associates (1965-7) working with Anabaenopsis raciborskii Wolosz., Cylindrospermum majus Kiitz, and a species of Anabaena, observed differences in the responses of the algae to different sources of combined nitrogen. For example, nitrate and nitrite did not completely inhibit heterocyst formation in Anabaena sp., whereas ammonium chloride did. I n A. raciborskii and C. majus, on the other hand, neither ammonium, nitrate nor nitrite inhibited it completely; though they extended the duration of the exponential growth phase, inhibited akinete formation and caused partial lysis at the end of the growth period. I n Anabaena doliolum all three sources inhibited heterocyst formation, though the degree of effectiveness was characteristic of each source (Singh & Srivastava, 1968). At a concentration of 0.02 M, nitrate completely eliminated heterocysts, whereas ammonium and nitrite caused a significant decrease in their frequency without completely inhibiting them. Cultures grown under the inhibitory influence of combined nitrogen (inhibition medium), not only lack heterocysts but possess cells of small size and a higher pigment content. However, heterocysts appear when the undifferentiated cultures are given a medium lacking combined nitrogen (hereafter called nutrient shift) (Talpasayi & Kale, 1967; Kulasooriya et al., 1972). Differentiation of heterocysts initiated in nitrate or ammonia-grown non-heterocystous cultures is completed within 12 h in Anabaena doliolum (Tyagi, 1 9 7 3 ~and ) about 24 h in A. variabilis (Ogawa & Carr, 1969). T h e heterocysts thus formed manifest a constancy in situation and steadiness in frequency. However, in contrast to the sequential development (and its dependence on vegetative growth) in nitrogen-fixing conditions heterocyst differentiation under nutrient shift is a simultaneous process (several heterocysts develop with characteristic cellular spacings between them). Furthermore, during the time that it takes for the heterocysts to develop to maturity under nutrient shift, no appreciable increase in growth by cell division takes place (Tyagi, 1973a), i.e. the period of differentiation is less than the generation time of the alga (see Wilcox, 1970). T h e origin of such a pattern of heterocysts has been attributed to the existence of nutritional gradients in the filaments (Fogg, 1949; Fritsch, 1951). Fogg (1949)

V. V. S. TYAGI postulated that the heterocyst is formed from a cell in the growing filament where the concentration of a specific inhibitory substance, probably ammonia or a derivative, falls below a critical level. At a point midway between two heterocysts inhibitor concentration will be least (assuming that the heterocysts secrete the inhibitory substance, see p. 273), and the cell here would be the most likely candidate for becoming a heterocyst. Fritsch (1951) put forward a similar hypothesis suggesting that certain growth-stimulatory substances (which could be derivatives of ammonia) are released into vegetative cells from heterocysts, and the continuous growth of vegetative cells decreases the concentration of these substances between two heterocysts. A new heterocyst would form in the centre when the concentration had decreased sufficiently. This implies that heterocysts prevent neighbouring cells from differentiation into heterocysts. That such a condition actually exists was demonstrated by Wolk (1967) who found that physical separation of vegetative cells from heterocysts decreased the growth rate of these vegetative cells and that enhanced heterocyst formation occurred after such fragmentation. In cultures of Anabaena grown in the presence of ammonia some incipient heterocysts or proheterocysts have been observed (Talpasayi & Kale, 1967; Talpasayi & Bahal, 1967; Wilcox, 1970). These are somewhat enlarged cells which are otherwise similar in shape, size and pigmentation to vegetative cells, and are distributed with almost regular cellular spacings in the filament. Kale & Talpasayi (1969) suggested that preheterocysts are cells which fail to transform into heterocysts in the inhibition medium but do so in the ammonia-free medium. Wilcox (1970) observed by phasecontrast microscopy, a regularly spaced pattern of proheterocysts in Anabaena cylindrica grown in the inhibition medium, and that the proheterocysts transform into heterocysts on nutrient shift. These observations suggested that ammonia or a derivative cannot be the only morphogenetic substance controlling heterocyst formation, but still provides for the possibility, as Fogg et al. (1973) point out, that its endogenous level can inhibit the differentiation up to proheterocysts. In an attempt to explain the regular spacing of proheterocysts Kale & Talpasayi (1969) suggested that only certain cells or ‘potential cells’ possess the genetically determined ability to develop into proheterocysts and subsequently into heterocysts. Later, Bahal & Talpasayi (1972) proposed a hypothetical model with a different notion to explain the pattern. They presumed that a differential growth pattern in the filament (faster near the heterocyst and slower in the middle) brings about the depletion of the middle cells owing to the flow of material (including nitrogenous) towards the areas of active division. Consequently, the middle cells achieve the minimum intracellular level of nitrogen and begin to differentiate into proheterocysts. This cycle is repeated as the filaments grow and results in the regular distribution of heterocysts in a medium free of combined nitrogen. In the inhibition medium, the proheterocysts and neighbouring cells divide more rapidly than others, thus causing the polarized flow of nutrients towards them, which would favour proheterocyst formation in the nutritionally depleted cells. This explanation may appear logical but suffers from the lack of proof that such a differential division actually occurs and that the gradient of nutrients exists. Moreover, differential division is guided by a nutritional (chiefly


259

nitrogen) gradient, and its occurrence when the nitrogen supply is abundant (in inhibition medium) is not understood. W'ilcox et al. (1973 a ) have studied heterocyst formation in Anabaena cylindrica and A. catenula and described the sequence of events in the determination of the pattern. These workers agreed that heterocysts produce an inhibitory substance, which diffuses into the vegetative cells and is possibly destroyed by them, so that a gradient is established around a heterocyst. There is a threshold level of the inhibitor below which differentiation begins (the above-threshold region defining an inhibitory zone). Cell divisions are asymmetrical, and in the sub-threshold region only the smaller daughters of such a division can transform into heterocysts (Mitchison & Wilcox, 1972); this provides a mechanism for limiting the number of potential heterocysts in this region. That close pairs of heterocysts do not arise is also due to a competitive interaction between several proheterocysts in the sub-threshold region (the authors have also used the term proheterocyst to distinguish the earliest stage in heterocyst differentiation in a medium free of combined nitrogen). That such a competition may be occurring is indicated in certain cases on nutrient shift. In ammonia-grown filaments, groups of 2-8 proheterocysts, besides the usual isolated proheterocysts could be seen. I n the ammonia-free medium this group pattern is resolved into a distinct pattern in which generally single proheterocysts develop into heterocysts ; the rest assuming the appearance of normal vegetative cells and going into division. Working on the principle that if a proheterocyst inhibits the development of adjacent proheterocysts it should also inhibit its own, Wilcox et al. (1973 a, b) provided further evidence for such a competition under normal growth conditions by breaking the filament at selected points next to, or a few cells away from, an early proheterocyst. Breaking the filaments to leave one cell next to the proheterocyst resulted in the regression of the proheterocyst to a vegetative cell stage in about 75 yo of cases. When two cells were left, proportionally fewer regressions occurred and with three cells the frequency of regression was insignificant (Wilcox et al., 1973a, b). T o confirm that such regression is not non-specific but is a part of the pattern-forming process, it was shown that the amino-acid analogue 7-azatryptophan significantly reduced the frequency of regression and close pairs of heterocysts developed in its presence (see also Mitchison & Wilcox, 1973). T h e authors have accounted for the regression of proheterocysts as the inhibitory effect of a cell upon itself and envisaged that supporting vegetative cells are required for mitigating the inhibitory effect. After eliminating the possibility that the regressions may be caused by the inhibitory effect of a neighbouring heterocyst, Wilcox et al. ( 1 9 7 3 ~ )interpreted the regressions by assuming that the proheterocyst synthesizes an inhibitory substance and is susceptible to its effect. If the concentration of this inhibitor within the proheterocyst exceeds a certain critical level, the proheterocyst begins to regress. This critical level rises as the proheterocysts develop, while at the same time the inhibitor is produced at an increasing rate. T h e cell continues to develop only if the inhibitor concentration within it does not rise above a critical level; therefore the inhibitor must be destroyed (or lost) from vegetative cells if a stable gradient is to be set up. T h e effect of two or more sources of inhibitor at a given cell is simply the sum of their individual contributions.

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Thus, if two proheterocysts begin to develop close together each will cause an increased level of inhibitor in the other by its contribution. Within a critical distance only one proheterocyst will be able to develop. At present, the interpretation of Wilcox et al. (1973a) seems to be a convincing explanation for the formation of an intercalary pattern. This envisages two morphogenetic substances of different chemical nature in contrast to one as postulated by Fogg (1949).One morphogen might be ammonia or a derivative which defines the inhibitory zone around the heterocyst. The second morphogen believed to be produced by the proheterocyst cannot be ammonia since proheterocysts (heterocysts without 3 layers of outer wall) have been shown to be unable to fix nitrogen (Kulasooriya et al., 1972 and see p. 251).Moreover, if this is ammonia one should not expect any proheterocysts in the inhibition medium (compulsory regression having occurred in this case). It is possible that at the sub-threshold level of inhibitor from the heterocyst a background concentration of a non-diffusible inducer (called X by Wilcox et al., 1 9 7 3 ~is) present and this is converted gradually into the hypothetical diffusible inhibitor (the second morphogen, Y). The relative proportions of X and Y in a cell would define its state of development. The cell with least initial Y would be the most suitable candidate for becoming a heterocyst and the smaller daughter of an asymmetrical division in the subthreshold region seems to fulfil this requirement (if the efficiency of a cell as a source of inhibitor is an increasing function of its size). With the revelation of the nature of X and Y and the control systems regulating their endogenous level, our knowledge of heterocyst formation would be much more complete. However, the model must also explain the nature of determinants in the regularly spaced pattern of proheterocysts in the inhibition medium where the inhibitory zones seem to be absent and all the cells in a filament may be supposed to have a similar background level of X. That the gradients determine the site of heterocyst formation under normal growth has also been observed in another genus, Cylindrospermum which, unlike Anabaena, contains only two terminal heterocysts (Reddy & Talpasayi, 1974).When either of the heterocysts is detached, regeneration of a new heterocyst occurs opposite to the end containing a heterocyst and after the attainment of a definite vegetative growth. I n filaments with both heterocysts detached, a heterocyst would form first at the end which lay farthest from a heterocyst in the intact filament and secondly after some increase in length. If heterocysts produce an inhibitor, its concentration in a vegetative cell would be proportional to the distance of that cell from the heterocyst, and the cell with least amount of inhibitor would undergo transformation. Further evidence that the endogenous inhibitor emanating from the heterocyst is a nitrogenous substance has come from determinations of the carbon: nitrogen ratio at various stages of differentiation. Kulasooriya et al. (1972)have found that the differentiation of heterocysts in undifferentiated (ammonia-grown) filaments of Anabaena cylindrica is regulated by the endogenous ratio of C:N. Proheterocysts were not detected when the C:N ratio in filaments was $ 5 : I , but they appeared as the C: N ratio increased to 6: I . With the advancement in heterocyst development the ratio also increased and finally stabilized at 8 : I ; at this stage the differentiation of the


26 I

Light

Asvmrnetrical cell division

I

ATP

I

‘>CYST]

Combined nitrogen inhibition

L PHASE I

Sub-threshold level of inhibitor or a specific C: N ratio REQUIREMENTS

PHASE II Protein synthesis

I

Carbon skeletons REQUIREMENTS

Fig. 2.A partially hypothetical scheme of stages and requirements of heterocyst differentiation. Phases I and I1 are continuous in the medium free of combined nitrogen, but in the inhibition medium differentiation does not proceed beyond phase I.

heterocyst was complete. In normal growth conditions a similar situation can be envisaged where a new heterocyst develops from a cell lying between two existing heterocysts and having the characteristic C :N ratio. How does combined nitrogen in the medium or endogenous nitrogen under normal growth conditions inhibit heterocyst formation? Neither the mechanism nor the interactions at chemical level are yet known. It is, however, amply evident that combined nitrogen inhibits reactions in the morphological differentiation of proheterocysts. Therefore, it can be envisaged that the necessary genetic information is generated during cell division in the inhibition medium and is conserved in the proheterocysts (this does not mean that a single cell is genetically determined), but its further expression remains inhibited till the arrival of appropriate conditions. Removal of the sources of combined nitrogen provides the so-called conducive physiological environment and switches on the differentiation process. In terms of the interactive model of Wilcox et al. (1973a) we can say that at this stage proheterocysts begin to produce the hypothetical inhibitor and compete with neighbouring cells (as in groups of proheterocysts on nutrient shift). Besides, chemical changes leading to development occur simultaneously in the competent cell, and its full morphological capabilities are revealed in the absence of combined nitrogen (or presence of nitrogen gas). It can be safely proposed that the whole process of heterocyst formation occurs in two distinct phases : I, synthesis and conservation of essential macromolecules during the phase of growth, and 11, activation of those macromolecules leading to the appearance of morphologically distinct heterocysts: this second phase is independent of growth.

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V. V. S. TYAGI

Phase I corresponds to proheterocyst formation in the inhibition medium and phase I1 to heterocyst differentiation under nutrient shift (Fig. 2). During growth in combined nitrogen-free medium phase I1 readily succeeds phase I. This succession is so quick that the two appear almost inseparable and the whole process seems to be continuous, Synthesis of macromolecules (DNA and probably mRNA) in phase I is integrated with cell division (asymmetrical) and is controlled by it, whereas activation and expression of the molecules (protein synthesis and morphological differentiation) in phase I1 has its own rate independent of growth by cell division and seems to be controlled by interactions between proheterocysts. This view is supported by the fact that heterocyst development under nutrient shift is closely paralleled by the appearance of nitrogenase activity (Kulasooriya et al., 1972). This analysis is also in agreement with Fogg’s (1949) view that heterocyst formation is affected by (i) rate of growth in cell numbers and (ii) rate of heterocyst differentiation, the two corresponding to the above-mentioned phases I and I1 respectively. Further evidence (in later sections) indicates that proheterocyst development in phase I1 is also controlled by environmental factors and has complex physiological requirements. Variations in frequency of heterocysts caused by environmental and nutritional factors in normal growth may be explained by the effects of these on either or both of the phases. Normally heterocyst frequency is determined by the close similarity in the reaction rates of the two phases. But a substance stimulating only the cell division (other than nitrogen or carbon source) could decrease heterocyst frequency by increasing the products of phase I (or proheterocysts) without simultaneously increasing the rate of heterocyst differentiation or phase 11. T h e reverse would be true for a growth inhibitor which might cause a transient increase in frequency by inhibiting cell division but not the development (phase 11) of any proheterocysts. Ultraviolet irradiation and substances such as the amino-acid analogue 7-azatryptophan seem to increase the frequency by affecting the mechanism of competitive interaction between proheterocysts in phase I1 only (i.e. they have no effect on growth or phase I). By way of analogy, the case of root nodules may be considered here. T h e formation of nodules is inhibited by enriching the soil with sources of assimilable nitrogen such as those employed in the inhibition medium of the blue-green algae (see Stewart, 1966). The nodules, nevertheless, regenerate on removal of the nitrogen sources, and the rate of differentiation does not bear any significant relationship with the growth of the plant (see Bergerson, 1969). T h e nitrogen compounds in this case obviously control differentiation at the level of morphological expression but the exact mechanism is again not known. Stewart, Fitzgerald & Burris (1968) and Ohmori & Hattori (1971, 1974) have shown that combined nitrogen inhibits nitrogenase synthesis in Nostoc muscorum and Anabaena cylindrica. How heterocyst formation is linked with nitrogenase synthesis is again not understood at present. Singh (1969) believed that control of heterocyst formation by nitrogen sources is exercised at the level of enzyme synthesis. He supposed that the inhibition of heterocyst (or proheterocyst ?) development and of nitrogenase synthesis by combined nitrogen is simultaneous because their multicistronic operons are controlled by the same regulatory gene. Combined nitrogen acts as a repressor and switches off both the processes, whereas in the presence of


263

molecular nitrogen (inducible condition) both the operons are switched on, leading to nitrogenase synthesis and heterocyst formation. This model is fascinating but suffers from a lack of unequivocal evidence in its favour.

( b ) Photoregulation Fogg (1949) observed an increase in heterocyst frequency in Anabaena cylindrica under feeble illumination but it is not clear whether this was caused by the effect of light on growth or more directly on heterocyst formation. However, it opened new vistas in research on the photoregulation of heterocyst formation. Lazaroff & Schiff (1962) and Holohan & Moore (1967) established definite photic requirements for growth and development including heterocyst formation in Nostoc muscorum. The morphogenetic events in this alga were believed to be controlled by light in a way similar to the photoperiodic control of flowering in higher plants (Lazaroff, 1966). Recently, differentiation of heterocysts in non-heterocystous filaments caused by nutrient shift has been used as an experimental system for the study of the effects of various factors including illumination on heterocyst development. Kale & Talpasayi (1969) and Kale (1972) established that light was essential for heterocyst development in Anabaena ambigua. Red light (600-700 nm) was found to be stimulatory to heterocyst formation, whereas green (450-550nm) was completely inhibitory. Further, no heterocysts were formed in dark-incubated cultures; nor could a supply of glucose substitute for the effect of light. Also, the presence of carbon dioxide is necessary in addition to a minimum 6 h photoperiod. The frequency of heterocysts varied linearly with increasing light intensity, but a maximum was attained at 1300 lux. Similar results with respect to light intensity and quality were obtained in A. doliolum (Kaushik & Kumar, 1970). On the basis of her findings, Kale (1972) seems to argue for a photomorphogenetic nature of the control (flowering plant type) and also stipulates the involvement of a photoreceptive pigment. Although allophycocyanin is indicated as the photoreceptive pigment for red light induction in N . muscorum (Lazaroff, 1966) and a new phytochrome-like pigment (having absoption maxima in the green and red regions) in Tolypothrix tenuis (Scheibe, 1972), the possibility of heterocyst differentiation involving stimuli generated through these pigments seems very remote. The reasons against an exclusive photomorphogenetic control are : (i) whereas photomorphogenetic effects of light can occur at very low illuminations (ineffective in photosynthesis) heterocyst differentiation needs light of high intensity (e.g. a minimum of 700 lux in A . ambigua; Kale, 1972) and (ii) very short exposures to light induce morphogenesis (e.g. 20 min in experiments of Lazaroff, 1966)) but heterocyst formation requires long photoperiods for initiation (see Singh & Viswanathan, 1972). Further, an obligate requirement for carbon dioxide in the presence of light and a partial substitution of the latter by glucose indicate non-morphogenetic effects of light. These requirements of heterocyst differentiation suggest (i) the involvement in it of the products of photosynthesis and/or (ii) involvement of some other photoregulated mechanism. The attainment of the highest frequency of heterocysts in red light, followed by yellow and blue, and their complete inhibition in green light together with their maximum frequency in white light (Kaushik & Kumar, 1970; I7

BRE

50

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V. V. S. TYAGI

Kale, 1972) emphasize the resemblance between the photic conditions for heterocyst formation and those for photosynthesis. At the level of ultrastructure, light inaugurates a series of morphological changes of high importance in the differentiation of the heterocyst (Robinson & Miller, 1970). Within the dark-grown undifferentiated cultures of Nostoc muscorum some of the cells, though quite indistinguishable in the light microscope, have been identified as immature heterocysts in electron micrographs (Ginsberg & Lazaroff, 1973). Such heterocysts possess granular inclusions and are packed with thylakoids, but on illumination the granules gradually disappear and the membranes decrease centrifugally. The occurrence of immature heterocysts in the dark-grown cultures and their subsequent differentiation on exposure to light have also been observed in Chlorogloea fritschii (Whitton & Peat, 1967). T h e nature of such effects is not understood at present; it is, however, known that light activates certain enzymes and initiates the synthesis of others in green plants (Zucker, 1972). During heterocyst differentiation some enzymes must be activated (or synthesized) in order to break down the granular material and to synthesize the additional wall. Such effects of light may differ from those usually called photosynthetic. I n addition, light also generates, through photosynthesis, some ingredients, such as ATP, reductant and a carbon source, which are required for the metabolism of the heterocyst (Stewart et al., 1969). Thus the light seems to produce some effects through interaction with enzymes (nonphotosynthetic effects) and some directly through photosynthesis. If the ' heterocysts' of dark-grown cultures of N . muscorum and C .fritschii are considered to be equivalent to proheterocysts, then light may be assigned a key role in phase I1 of heterocyst differentiation. (c) Metabolic control Some studies have recently been undertaken on the metabolic control of heterocyst differentiation using specific metabolic inhibitors. T h e effects of the inhibitors could cause (i) the induction of lag in the appearance of heterocysts under nutrient shift, and (ii) a drop in heterocyst frequency, and both the parameters have been used. Bahal & Talpasayi (1970~) found that some thiol inhibitors such as mercaptoethanol, p-mercuribenzoate, iodoacetate, and ethyl maleimide significantly reduced heterocyst frequency. An inhibition of heterocyst formation by these inhibitors indicates the involvement of some SH-containing enzymes in differentiation. These studies, however, do not indicate the involvement of a specific metabolic pathway, nor do they identify the enzymes involved. Bahal & Talpasayi (197ob) further found a reduction in heterocyst frequency on addition of sodium azide or glycine to the medium. Glycine is used as a nutrient by A. cylindrica and inhibits heterocyst formation by acting as a nitrogen source (Fogg, 1949). T h e effect of azide, however, is manifold and remains ambiguous in the experiments of Bahal & Talpasayi. z,4-dinitrophenol, an established inhibitor of oxidative phosphorylation, has been stated to stimulate heterocyst formation (Bahal, 1969). Studies with some specific inhibitors of photosynthesis (e.g. 3,p-chlorophenyl-1,1dimethylurea (CMU)) and respiration (e.g. z,q-dinitrophenol (DNP), sodium azide, and iodoacetic acid) have been carried out on Anabaena doliolum (Tyagi, 1 9 7 3 ~ ) .

266

V. V. S. TYAGI

(i) Archaic reproductory structures Geitler (1921 a ) regarded the heterocysts as relic reproductive structures on the basis of the observations that these cells, like the akinetes, occasionally germinate to give rise to new filaments. The first case of heterocyst germination was probably found by Bornet & Thuret (1880)in Nostoc ellipsosporum (Desm.) Rabenh. Formation of endospores (which give rise to new filaments on germination) in the heterocysts of Anabaena cycadeae Reinke was noted by Spratt (191I) and subsequently in Calothrix (Steinecke, 1932), Rivularia (Desikachary, 1946), and in several other genera (see Kale & Talpasayi, 1969) germinating heterocysts were found. Recently, some workers have induced germination in heterocysts in controlled conditions. Wolk (1965b) discovered germination of heterocysts of A. cylindrica, although at low frequencies (maximum IO%), in a culture medium supplemented with glucose (50 mM/1) and ammonium chloride ( 4 0 mM/l). Singh & Tiwari (1970)have also been able to induce about 5 % germination by treatment with ultraviolet light in Nostoc linckia (Roth) Born. & Flah. - a species never showing it under natural conditions. Furthermore, 24% germination was recorded by these workers in a mutant form of this species, and up to 83 yo in a non-sporulating mutant strain of Gloeotrichia ghosei Singh (Singh, Tiwari & Singh, 1972). Morphological stages in germinating heterocysts in these cases were found similar to akinete germination under natural conditions. These observations seem to support the view of Geitler (1921a) that the heterocysts were developed for reproduction and have lost this function in course of time. Sometimes, however, they revert to their original function. Fritsch (1951)disagreeed with this view on the basis that the akinetes are reproductory bodies in the majority of heterocystous blue-green algae and that it would be of no advantage to the algae to develop two structures with similar function. The heterocysts also lack such features as the presence of reserve material and a resistant envelope characteristic of spores. Nevertheless, the phenomenon is indicative of the totipotency and physiological plasticity of heterocysts, although this plays little part in the life cycle of the algae in natural habitats. Wilcox et al. (1973b) have found that when heterocysts at certain stages of their development are detached from the filament, they show signs of regression to the original stage of development. While fully mature heterocysts do not regress, those doing so exhibit division of protoplast and ‘casting off’ of the envelope. Thus these authors considered germination as a case of reversion of proheterocysts to the vegetative cell stage, and further pointed out that in early observations the origin of the germling was not ascertained, i.e. whether from a regressing proheterocyst or from a mature heterocyst. The role of ultraviolet light and ammonium ions in the induction of germination is not understood. The frequency of germinating heterocysts in the presence of ammonia is usually low, so that any hypotheses to explain the action of ammonia must account for its inability to induce germination in the majority.

The heterocysts of blue-green algae (Myxophycae)

265

CMU, DNP, azide and iodoacetic acid inhibit heterocyst formation effectively and the inhibitory effect of CMU is reversed by glucose while the DNP-inhibition is not; A T P causes a partial reversal in the latter case. From this study of inhibitors it may be concluded that heterocyst formation in this alga depends on the availability of carbon intermediates and ATP; the former are supplied by photosynthesis and ATP most probably by oxidative metabolism. T h e roles of protein synthesis and nucleotide metabolism in heterocyst formation are not precisely known. Heterocyst formation is inhibited by chloramphenicol (Talpasayi & Kale, 1967) and by streptomycin (Kumar & Kaushik, 1971) indicating that protein synthesis is involved. Unequivocal evidence on the biosynthesis of specific proteins is, however, lacking. Similarly, nothing can be said with confidence on the role of nucleic acids and the control of protein synthesis. T h e fact that heterocyst formation on nutrient shift occurs in a period less than the generation time of the algae does not suggest an immediate role of the nucleic acids. I n addition, the failure of mitomycin C (an established inhibitor of DNA-dependent D N A synthesis in bacteria) to inhibit heterocyst differentiation indicates that replication of the genome does not occur before heterocyst formation (Tyagi, 1975). Also actinomycin D (an inhibitor of DNAdependent RNA synthesis) inhibits RNA synthesis in A. doliolum without significantly inhibiting heterocyst formation (Tyagi, 1973 b). T h e main contribution of the studies with inhibitors of nucleotide metabolism is that heterocyst formation under nutrient shift does not involve immediate D N A replication or transcription. It seems that the genome is duplicated, and the formation of information carriers all takes place during growth in the inhibition medium, i.e. in phase I of heterocyst differentiation. Probably the proheterocyst contains the replicated genome and possibly the other intermediates (e.g. mRNAs) also. On the approach of conducive conditions, these cells immediately embark upon differentiation and form distinct heterocysts -the process involving activation and expression of the macromolecules - corresponding to the phase I1 of heterocyst differentiation. During growth in nitrogen-fixing conditions, phase I1 follows phase I so quickly as to present an integrated pattern. Furthermore, the fact that streptomycin (translational inhibitor of protein synthesis) inhibits heterocyst formation, whereas actinomycin-D (transcriptional inhibitor) does not, is important in the contest of our present knowledge of the control of protein synthesis in blue-green algae and prokaryotes in general. V. FUNCTIONS

Notwithstanding our ignorance of the details of biochemical pathways in the heterocyst, it is fairly understood that this cell is metabolically active and its existence of vital importance to the algae. T h e question of heterocyst function is an interesting story of research and is also the cause of current interest in this cell. Much work has been done on this aspect since the comments of Fritsch ( I ~ s I ) ,and in accordance with its metabolism physiological roles for the heterocyst have been proposed (see Fay, 1973). This section deals with various functions ascribed to the heterocyst from time to time and analyses their validity in the present context. 17-2


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(ii) Storage cell Hieronymus (1892) and Fritsch ( 1 9 0 4 ~proposed ) that heterocysts are the storage vessels of cyanophycin when it is overproduced in the adjacent vegetative cells. However, the absence of any large quantity of reserve food in the heterocyst seems to speak against this view. Canabaeus (1929) considered that the heterocysts are storehouses of enzymic substances and promote the growth of the algae by secreting these substances into the adjacent cells. The presence of enzymes was not, however, demonstrated by Canabaeus and her suggestion has not been generally accepted. Nevertheless, Fritsch (1951) derived a new concept from these observations and postulated that primarily the terminal heterocyst of the filament secretes substances which promote growth and cell division. As cells become farther removed by growth and cell division from the existing heterocysts, additional centres of enzyme activity become necessary and intercalary heterocysts are formed. The hypothesis of Fritsch necessitates that the ‘growth promotory substances’ should either be synthesized in the heterocyst or some precursors migrate into them from adjacent cells. Recent work has broadly supported the hypothesis of Fritsch, and the current opinion is that the heterocysts rapidly pump into the vegetative cells some essential substances needed for the active physiological state of these cells. The phrases ‘centres of enzyme activity’ and ‘storehouse of reserve food’ may now be applied to heterocysts, but in a modified sense and not literally as originally used. The synthesis and release of vital substances into adjacent cells seems to be the main metabolic activity of the heterocysts but the details are subjects of real concern at present. (iii) Attachment organ DePuymaly (1957), suggested that heterocysts serve as organs of attachment for the developing trichomes. This hypothesis has been recently revived by Allsopp (1968), who noted that during development the hormogonia in the hormocysts of Scytonema javanicum (Kutz) Born. generally produce a central heterocyst. This heterocyst remains attached to the sheath of the hormocyst and functions as an organ of attachment for the new filaments to the sheath. Stewart (1972), however, pointed out that such a function may be important only in terrestrial members of the Scytonemataceae and Stigonemataceae and may not be so relevant in planktonic genera such as Anabaena, Rivularia and Gloeotrichia. Moreover, little experimental evidence has so far been provided in favour of such a role. (iv) Regulation of akinete formation (sporulation) The akinetes (spores) of heterocystous blue-green algae are formed in specific spatial relation to the heterocyst (see Fritsch, 1945; Desikachary, 1959). In genera such as Gloeotrichia and Cylindrospermum the only akinete differentiates in immediate vicinity to the single terminal heterocyst, whereas in Anabaena cylindrica akinete formation begins near the heterocyst and gradually proceeds toward the middle of the filament. Such instances are indicative of some interaction between vegetative cells and heterocysts, with the latter seemingly inducing sporulation

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(Fritsch, 19oqb; Geitler, 1921b). Some workers observed a gradual depletion of the contents of heterocysts during sporulation and interpreted it as denoting a function of supplying material needed in spore formation (Bharadwaja, 1933; Singh, 1942). More recently, Wolk ( 1 9 6 5 ~ has ) found that spore formation in Anabaena cylindrica begins adjacent to the heterocyst and gradually progresses away from it. Wolk (1966) believes that sporulation of a vegetative cell near the heterocyst in A. cylindrica depends upon (i) a sequential process beginning near the heterocyst and (ii) mitigation of ' a sporulation-inhibitory influence ' acting on the vegetative cells. Additional evidence implicating heterocysts in sporulation came from the observations that differentiation of cells adjacent to a heterocyst is prevented by separating the two cell types, but without apparent loss of differentiation potency of these cells (Wolk, 1966). Despite much convincing evidence in favour of an inductive role of the heterocyst in sporulation, some doubts are raised about its wide applicability. Spores occur in characteristically different positions in relation to the heterocyst in other genera ; for example, in Anabaena laxa (Rabenh.) A.Br. they are formed near heterocysts but not just adjacent to them (Canabaeus, 1929). Sporulation in A . doliolum begins half-way between two heterocysts and proceeds centrifugally towards the heterocystous ends, and a few cells nearest to the heterocyst may fail to sporulate. These facts actually suggest an inhibitory role of the heterocyst in sporulation in this alga. Further, critical levels of carbon and nitrogen are involved in spore differentiation in A . doliolum and the heterocyst probably regulates these factors through its own metabolism (Tyagi, 1974b). Singh & Srivastava (1968), on the basis of their observations that certain concentrations of potassium nitrate completely inhibit sporulation but not heterocyst formation in A . doliolum, suggested that the two phenomena are not related. However, the details of the physiology of akinete formation in both kinds of algae, viz. those forming akinetes adjacent to a heterocyst and others away from it, are required in order to reach a conclusion in this regard. Some other functions have been assigned to heterocysts by various workers. Troitzkaia (1924) observed an enlargment of cells adjacent to heterocysts in a species of Anabaena and supposed that these cells produce substances which cause an increase in the volume of vegetative cells. It has also been proposed that heterocysts induce synthesis of walls in dividing cells in A. axollae, and that in Tolypothrix tenuis the heterocyst can cause an adjacent cell to transform into a heterocyst (Schwabe, 1947a, b). A secretory role for the heterocyst was envisaged by Tischer (1957) based on the finding that bacteria accumulate near the heterocyst. A function in fragmentation of filaments and hormogonia formation was considered earlier by Borzi (1878) and was subsequently upheld by Geitler (1921b). This is, however, a fortuitous or incidental function.

(w) Nitrogen jixation The blue-green algae share with bacteria the property of fixing atmospheric nitrogen. A number of these algae have been examined and now about 50 species are known to be active nitrogen fixers (Stewart, 1970; Fogg et al., 1973). With the exception of a few


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sporadic genera, the nitrogen fixers belong to the Nostocales and the Stigonematales and are generally the filamentous forms possessing heterocysts. These observations together with experimental findings on the localization of reducing activity led to the origin of the belief that the nitrogen-fixing system of the blue-green algae is sited in the heterocyst (Fay et al., 1968). This has been followed by several convincing arguments, particularly from the laboratory of W. D. P. Stewart at Dundee, Scotland. Nitrogen fixation was first demonstrated in two heterocystous genera - Nostoc punctiforme (Kutz) and Anabaena sp. (Drewes, 1928). Other common genera which fix nitrogen are : Aulosira, Cylindrospermum, Tolypothrix, Calothrix, Scytonema, Stigonema and Hapalosiphon - all having heterocysts. Chlorogloea fritschii, a species earlier described as a unicellular, heterocystless nitrogen fixer (Mitra, 1950; Fay & Fogg, 1962) has now been found to be a filamentous form resembling Nostoc and producing characteristic heterocysts (Fay et al., 1964; Stanier, Kunisawa, Mandel & Cohen-Bazire, 1971). Similarly, Aphanizomenon Jos-aquae (L.) Ralfs. a characteristically heterocystous alga, was described earlier as unable to fix nitrogen (Williams & Burris, 1952). There is now evidence both from the laboratory (Stewart et al., 1968) and the field (Mague & Burris, 1973) that this alga fixes nitrogen although it possesses relatively very few heterocysts. A direct relationship between heterocysts and the capacity to fix nitrogen has also been demonstrated in some common blue-green algae (Jewel1 & Kulasooriya, 1970). Any attempt to generalize the relationship between the possession of heterocysts and the capacity to fix nitrogen is, however, prevented because there are reports in the literature of nitrogen fixation by non-heterocystous algae. Nitrogen fixation has been observed at significant rates in the filamentous marine alga, Trichodesmium (Osciliatoriaceae) (Dugdale, Goering & Ryther, 1964). Goering, Dugdale & Menzel(1966) and Dugdale & Goering (1967) have detected high rates of light-dependent nitrogen fixation in this alga using the heavy isotope l6N, and Bunt et al. (1970) have demonstrated nitrogen fixation in axenic cultures of this alga by acetylene-reduction technique. Stewart & Lex (1970) have shown nitrogen fixation under microaerophilic (N,/CO,) conditions in another non-heterocystous alga, Plectonema boryanum (Scytonemataceae). Nitrogen-fixing ability in other filamentous non-heterocystous genera such as Lyngbya aestuarii Liebm. was indicated earlier (van Baalen, 1962) and has been recently confirmed in several strains of Oscillatoria (Kenyon, Rippka & Stanier, 1972). Moreover, some unicellular genera of the Chroococcales have also been observed to possess nitrogen-fixing ability; for example, in cultures of Chroococcus rufescens Nag it was indicated by Cameron & Fuller (1960). More recently, Wyatt & Silvey (1969) and Rippka, Neilson, Kunisawa & Cohen-Bazire (1971) working independently with two different strains of Gloeocapsa, have found significant rates of acetylene reduction. These findings indicate that the nitrogen-fixing ability of the blue-green algae is not confined to the heterocystous genera only. However, from further evidence (seep. 271) it would appear that the heterocyst is the most suitable site for nitrogen fixation at least in heterocystous algae, although in other genera the activity may develop in vegetative cells under conducive conditions. On the other hand, there are mutants of Nostoc linckia and Anabaena doliolum which have been

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isolated after mutagenic treatments, and these possess heterocysts but lack nitrogenfixing ability (Ahmad & Venkatraman, 1971; Sinha & Kumar, 1973). These mutants may be useful in studies on the control of nitrogen fixation and heterocyst formation, but they scarcely prove that heterocysts normally are not associated with nitrogen fixation. The second line of evidence in favour of the view that heterocysts are involved in nitrogen fixation emanates from the observations that a supply of combined nitrogen in the growth medium inhibits heterocyst formation (Fogg, 1949; Stewart, 1964; Mickelson et al., 1967). Applying the teleological principle that the organs not required under a set of conditions tend to disappear, it strikes the imagination that the combined nitrogen possibly does away with nitrogen fixation and consequently with the heterocysts. Substantial evidence is now available that combined nitrogen inhibits the synthesis of nitrogenase in blue-green algae (Stewart et al., 1968; Bone, 1971; see also Streicher, Gurney & Valentine, 1972). The mechanism linking both these processes, however, remains to be elucidated. The nitrogen sources do not affect the activity of preformed nitrogenase ; neither do the mature heterocysts dedifferentiate on the addition of these substances (see Fogg et al., 1973). Further evidence implicating the heterocyst in nitrogen fixation is collated from the physiological and spatial relationship between heterocysts and akinetes. T h e akinetes in Anabaena cylindrica originate near the heterocyst and their formation is stimulated by the presence of ammonia in the growth medium (cited in Fay et al., 1968). It indicates that the site of akinete formation is the region of highest ammonia concentration. Several genera, such as Wollea, Cylindrospermum, Gloeotrichia, and Anabaena fyengari also form akinetes in close proximity to the heterocyst (Desikachary, 1959). Further, the inception of sporulation near the heterocyst and its centripetal advancement in certain species of Anabaena and Nostoc is indicative of the existence of an ammonia gradient beginning from the heterocyst (Wolk, 1965a). Nevertheless, all heterocystous algae do not form akinetes in close proximity to the heterocyst; this indicates that conditions for sporulation differ in various species, and no generalized conclusion should be drawn at present from it regarding heterocyst function. I n an otherwise complete but combined-nitrogen-deficient medium the growth of a nitrogen-fixing alga would be regulated by its capacity to fix nitrogen. The filaments of some nitrogen-fixing genera such as Gloeotrichia, Rivularia and Calothrix have characteristically whiplike shape (tapering into a colourless multicellular hair) with a single terminal heterocyst at the broader end. Such a structure suggests that growth of the filaments depends upon substances emanating from the heterocyst, and that the substances get diluted as they traverse from cell to cell. T h e observations of Walsby & Stewart (cited in Fay et al., 1968) that on culturing the alga in presence of ammonium chloride the whiplike shape is lost and the filaments exhibit uniformity in cell shape, argues for the ammoniacal nature of substances arising from the heterocyst under nitrogen-fixing conditions. Some evidence involving the heterocyst in nitrogen fixation is also provided by the fact that in heterocystous genera the first heterocyst differentiates immediately on germination of the spore in a medium deficient in combined nitrogen. Existence of the heterocyst would be essential in order to supply nitrogen to


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the growing filament under such conditions. Fay et al. (1968) and Singh & Srivastava (1968) have presented photographs of spore germlings at the 4-to ro-celled stage of Anabaena possessing a heterocyst. T h e problem of nitrogen fixation by heterocysts has been recently approached by examining the structural and metabolic suitability of this cell as a site of nitrogen fixation. I n bacteria, nitrogen fixation depends upon the availability of (i) ATP, (ii) an electron donor (reductant), (iii) some enzyme cofactors and (iv) carbon skeletons. Further, it is a reductive process and is inhibited by high atmospheric levels of oxygen (Burris, 1969; Stewart & Pearson, 1970). T h e blue-green algal nitrogenase in cell-free extracts also shows an absolute requirement.for ATP, a source of reductant and of cofactors (Smith & Evans, 1970; Haystead, Robinson & Stewart, 1970). T h e oxygen sensitivity of algal nitrogenase has also been established (Fay & Cox, 1967; Stewart & Pearson, 1970; Haystead et al., 1970). Therefore, in the blue-green algae which evolve oxygen during photosynthesis a strongly protected site is necessitated for the nitrogenfixing system. Ample evidence is available that heterocysts lack the oxygen-evolving photosystem I1 of photosynthesis (Bradley & Carr, 1971; Donze et al., 1972) and are thus suitable sites for harbouring the nitrogenase. Moreover, highly reducing conditions have been indicated by reduction of triphenyl tetrazolium chloride (TTC) in heterocysts (Drawert & Tischer, 1956; Tischer, 1957; Fay & Kulasooriya, 1972). Tetrazolium salts are readily reduced to water-insoluble formazan crystals and have been widely used to demonstrate the reductive capacity of cells (Drews, 1955). T h e majority of the vegetative cells show very little formazan, indicating that they are less reductive than the heterocysts (Talpasayi, 1967). Furthermore, higher respiratory activity has been shown in the heterocysts than in the vegetative cells (Fay & Walsby, 1966). Such a situation may be favourable to the oxygen-sensitive nitrogenase in that the internal oxygen tensions would be reduced by the high respiratory rate. T h e use of direct tests for nitrogenase activity in isolated heterocysts has been a more rewarding approach. Fay & Walsby (1966) made the first attempts of this type by using 15N-labelled nitrogen gas but could not find nitrogen fixation by isolated heterocysts of Anabaena cylindrica. Recently, however, Wolk, Austin, Bortins & Galonsky (1974) have shown by autoradiographic localization of 13N in intact filaments of A . cylindrica that about 25% of nitrogen fixation is carried out by the heterocysts. Stewart et al. (1969) successfully demonstrated nitrogenase activity by using acetylenereduction technique. T h e heterocysts reduced acetylene to ethylene in presence of (i) ATP - an energy source - and (ii) sodium dithionite as reductant. I n contrast to the observations of Stewart et al. (1969) Wolk & Wojciuch (1971a) presented evidence that isolated heterocysts reduce acetylene in the light in the absence of A T P and dithionite, although addition of these effectors enhances activity. These observations are significant since they offer direct evidence of acetylene reduction by heterocysts and also demonstrate that these cells can independently generate reductant in the light and can also photophosphorylate. Some workers have approached the problem by correlating the physiology of nitrogen fixation and the metabolism of the heterocyst. Cox & Fay (1969), working on the idea of a close relationship between nitrogen fixation and photosynthesis first

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envisaged by Fogg & Than-Tun (1960), found in Anabaena cylindrica that C M U (chlorophenyl dimethylurea) inhibits carbon dioxide fixation in the light but not nitrogen fixation and concluded that nitrogen fixation in this alga is independent of light-generated reducing power. Furthermore, increase in light intensity stimulated nitrogen fixation irrespective of the presence of CMU, thus indicating the involvement of light-generated ATP (in cyclic photophosphorylation in presence of CMU) in nitrogen fixation. The source of reductant was believed to reside in some dark reaction (see also Bothe & Loos, 1972). These aspects were elaborated by Fay (1970). Using monochromatic light he found that photostimulation of acetylene reduction occurs best at 675 nm, the wavelength most absorbed in pigment system I and little effective in oxygen evolution. These results suggested that photosystem I is involved in nitrogen fixation and its main function is probably to supply ATP through cyclic photophosphorylation. Such a scheme is in agreement with the findings that isolated heterocysts possess mainly the P,oo, the pigment of photosystem I ; they give low yield of chlorophyll fluorescence and photoreduction of P,oo,both indicating absence of functional photosystem 11 from them (Thomas, 1970; Donze et al., 1972). Working on the same lines, Lyne & Stewart (1973) have investigated the photostimulation of nitrogenase activity in A. cylindrica by comparing the effects of lights primarily absorbed by pigment system I and 11 on carbon fixation and nitrogenase activity. Red light ( > 660 nm) mainly absorbed by pigment system I supports acetylene reduction more efficiently, whereas the sodium light (589 nm) mainly absorbed by pigment system 11 supports carbon fixation. These observations corroborate the findings of Fay (1970) on the source of energy and implicate the heterocysts in nitrogen fixation. According to some evidence, the source of reductant, besides that of energy needed in nitrogen fixation, also resides in the heterocyst. On the basis of studies with whole cells and cell-free extracts two main views have been advanced regarding the nature of reductant. (i) Bothe (1970), Smith & Evans (1970), and Smith, Noy & Evans (1971) found evidence that reductant formation is light-dependent and that electron flow to nitrogenase is mediated by ferredoxin. Dichlorophenol indophenol and ascorbate in their experiments reduced nitrogenase in the light (indicative of electron transport through the photosynthetic transport chain). (ii) Cox & Fay (1967, 1969) found that pyruvate decarboxylation is coupled with nitrogen fixation and this occurs in a ratio of 3:1. They believed therefore that pyruvate acts as hydrogen donor for nitrogen reduction and the provision of reductant is independent of photosynthesis. I n a search for the source of reductant Lex, Silvester & Stewart (1972) have discovered that acetylene reduction is inhibited by 3-3,4-dichlorophenyl-1 ,I-dimethylurea (DCMU) under conditions stimulating photorespiration (high oxygen tension and low carbon dioxide concentration) but is unaffected under conditions which inhibit photorespiration. This suggests that the source of the reductant for both the processes is the same ; under low photorespiration the reductant accumulates and acetylene reduction is not immediately inhibited by DCMU. In this context, the inability of C M U to inhibit acetylene reduction in the experiments of Cox & Fay (1969) might be due to low rates of photorespiration by the alga. Lex & Stewart (1973) have further observed that monofluoroacetate, an inhibitor of aconitate hydratase activity in the Krebs cycle,


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inhibits actylene reduction to a greater extent in the dark than in the light, and D C M U and monofluoroacetate together can inhibit acetylene reduction completely in the light. It has been concluded by these workers that the reductant is supplied by a pool of carbon compounds and that photorespiration and nitrogenase activity compete for the reducing power. Under high photorespiration the pool size is small and acetylene reduction is inhibited by DCMU, whereas under low photorespiration the pool size is large and supplies reductant to nitrogenase even in the presence of DCMU. T h e inhibition of acetylene reduction by both monofluoroacetate and D C M U has been interpreted to suggest that the pool of carbon compounds furnishes electrons via the Krebs cycle and that photosystem I also acts as a mediator. Wolk (1968) presented autoradiographic evidence to show that the heterocysts receive photosynthates from the vegetative cells. I n this context, the above data of Stewart and coworkers suggest that, in the heterocyst, electrons may emanate from the photosynthates via the Krebs cycle and pass on to photosystem I and then probably to ferredoxin or some other specialized redox protein (Bothe & Loos, 1972; Haystead & Stewart, 1972) which transfers them to nitrogenase. Occurrence of high activities of the enzymes of the pentose phosphate pathway, e.g. glucose-6 phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and relatively low activity of ribulose-1 ,S-diphosphate carboxylase have been demonstrated in the heterocysts (Winkenbach & Wolk, 1973). These results suggest that heterocysts play a significant role in sugar dissimilation and generate reductant which might provide electrons to nitrogenase. Inability of heterocysts to carry on photosynthetic carboxylation indicates their dependence on the vegetative cells for photosynthates. Whether the sugars pass through the Krebs cycle or only through the oxidative pentose phosphate pathway to generate the reductant remains to be elucidated. The relative significance of the two pathways as sources of reductant can be evaluated on the availability of data on the enzymes of Krebs cycle in the heterocysts. Nevertheless, these results together with those of Van Gorkom & Donze (1971), Thomas & David (1972) and Wolk & Wojciuch (1971a, b) go a long way towards proving that the heterocysts are the most suitable sites for nitrogen fixation. T h e end-product of dinitrogen reduction in blue-green algae is probably ammonia, which is a potent inhibitor of nitrogenase synthesis (Ohmori & Hattori, 1974). Some enzymes have been detected in the heterocysts which scavenge the newly synthesized ammonia and convert it to non-inhibitory nitrogenous compounds (Scott & Fay, 1972). The activity of glutamine synthetase catalysing ATP-dependent amination of glutamate has now been demonstrated in the heterocysts of Anabaena cylindrica (Dharmawardene, Stewart & Stanley, 1972; Dharmawardene, Haystead & Stewart, 1973; see also Haystead, Dharmawardene & Stewart, 1973). These findings have led to the conclusion that besides reduction, the incorporation of ammonia into carbon skeletons also occurs in the heterocyst. Evidence demonstrating the presence in some algae of nitrogenase activity in the vegetative cells has appeared in recent years. Wolk (1970) was the first to raise such a possibility, and further evidence on this aspect was presented by Smith & Evans (1970). These workers demonstrated nitrogenase activity in extracts of cells of cultures,

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grown in N2/C02 environment and from which heterocysts (or vegetative cells) were isolated by means of the French press method. A requirement for ATP and dithionite was observed for nitrogenase activity. Stewart (1971, 1973) has criticized the inference of Smith & Evans in the light of the observations of Fay & Lang (1971) that during isolation by French press the heterocysts are partially damaged and the enzyme might have leaked out from them. This, however, does not exclude the possibility of actual existence of the enzyme in the vegetative cells of the alga grown in microaerophilic environment. Nitrogenase activity has also been demonstrated in anaerobically grown cultures of a filamentous but non-heterocystous alga, Plectonema boryanum (Stewart & Lex, 1970; Stewart, 1971).On transferring the ammonia-grown cultures of this alga to a microaerophilic environment the acetylene-reducing activity developed with a lag period. However, prior to the development of the activity, the phycocyanin :chlorophyll ratio was found to decrease significantly. Phycocyanin participates in oxygen evolution in blue-green algae, and the possibility that its absence under anaerobic conditions facilitates nitrogenase activity (since oxygen inhibits nitrogenase activity) has been envisaged (Stewart, 1971). The utilization of phycocyanin as nitrogen source during nitrogen starvation has also been shown (Allen & Smith, 1969; Neilson, Rippka & Kunisawa, 1971).Furthermore, a gradual decrease in phycocyanin fluorescence with increasing distances from the heterocyst under aerobic conditions and disappearance of this gradient under N,/C02 environment has been found (Van Gorkom & Donze, 1971). If abundance of phycocyanin is regarded as an index of the cell’s endogenous nitrogen reserve, the existence of a gradient of the kind found by these workers should imply the secretion of nitrogenous matter to adjacent cells from the heterocyst. Disappearance of the gradient in microaerophilic conditions suggests the development of nitrogenase activity in the vegetative cells. These findings together with the observations of Wyatt & Silvey (1969) and Rippka et al. (1971) on nitrogen fixation in Gloeocapsa, and of Kenyon et al. (1972) on OscilZatoria suggest that the nitrogenase activity in heterocystous algae is localized in heterocysts under aerobic conditions, although after incubation in an anaerobic environment the activity may develop in the vegetative cells also. Nitrogenase activity is irreversibly inhibited by high oxygen tensions in cell-free extracts of blue-green algae but not so in intact cultures (Stewart & Pearson, 1970; Haystead et al., 1970). Development of nitrogenase activity in a microaerophilic environment in vegetative cells and its absence from aerobically grown cultures raises the question of the state of nitrogenase in such cells. Oxygen is known to inhibit nitrogenase activity but not its synthesis (see Wong & Burris, 1972) and thus the presence of an active nitrogenase in the vegetative cells of heterocystous algae and of PZectonema is a strong possibility. High oxygen tensions probably inhibit heterocyst formation and it might be the cell’s internal oxygen tension which, together with the endogenous level of ammonia, that determines the activity and synthesis of nitrogenase in the prospective heterocyst.


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VI. GEOLOGICAL HISTORY

The fossil record shows that the heterocystous genera existed as far back as the Precambrian. The Rivulariaceae were probably the first to appear on the geological time scale (Archaeozoic; Korde, 1972) with Palaeorivularia and Jatulina as the first genera. The presence of heterocyst-like cells is indicated in three Middle Devonian fossils - Rhyniella (Scytonemataceae) and Kidstoniella and Langiella (Stigonemataceae) (Croft & George, 1959). Two other genera Anabaenidium and Archaeonema longicellularis presumably with heterocysts have been described from the Late Precambrian of Central and South Australia (Schopf, 1968; Schopf & Barghoorn, 1969). Sastri, Venkatchala & Desikachary (1972) have described from the Precambrian of India a genus named Palaeonostoc with distinct heterocysts almost as broad as the vegetative cells and about 9-10 pm long. The blue-green algae were probably the first oxygen-producing photoautotrophic organisms to evolve on the earth and their origin is coeval with the heterotrophic bacteria (Barghoorn & Schopf, 1966). The presence of a protected cell system to harbour an active nitrogenase must have been necessitated by a gradual disappearance of the reducing and the onset of an oxidizing atmosphere on the earth. This presumption is supported by experimental observations on the development of nitrogenase activity in the vegetative cells on exposure to oxygen-less atmosphere (Stewart, 1971). The presence in heterocysts of photosystem I and the absence of oxygen-evolving photosystem 11 also identify them with photosynthetic bacteria and are suggestive of their origin as an adaptive organ against an aerobic environment. Such an environment began to appear on the earth about 2.5 billion years ago and logically also the origin of heterocysts can be traced to the same period. The heterocystous algae as a whole may also be considered to have evolved as a symbiotic system between photosynthetic bacteria and oxygen-producing unicells. This system must have become integrated genetically and physiologically, and has been able to persist ever since.

VII. CONCLUSION

Evidence on the structural aspects of the heterocyst indicates the advanced nature of this cell. Its long evolutionary history suggests that it is probably the first case of morphological specialization in Prokaryota, and possibly in the plant kingdom. The evolutionary changes leading to the heterocyst may be reconstituted in the study of its ' ontogeny '. Obviously, the trend of evolution dating the formation of the heterocyst from the vegetative cells has not been all progressive. The loss of pigments and of granular substances might be considered steps in retrogressive evolution. Synthesis of an additional polysaccharide wall, the nitrogenase complex, and other yet unknown enzymes involved in wall synthesis are, however, definite cases of progressive evolution. The rearrangement of the thylakoids, disappearance of the usual lipids and the synthesis of a novel class in the heterocyst together with a different composition of the wall testify that heterocysts are a case of organizational modification evolved to fulfil some definite functions.

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With regard to the differentiation of the heterocyst some interesting aspects have been unravelled and others with a possible bearing on the phylogenetic relationship of Myxophyceae are likly to emerge. Is heterocyst differentiation of the kind met within the bacteria? Functional differentiation in bacteria is regulated through direct interaction of some component of the cell’s environment with the genome (Jacob & Monod, 1961; genetic operator model). I n the case of the green alga Acetabularia, on the other hand, the control of differentiation is thought to operate in the cytoplasm and not directly on the genome (Werz, 1969). T h e heterocyst can offer a model system for the study of this problem and others concerning cellular differentiation in Prokaryota. Elucidation of the control system of differentiation along with the mode of protein synthesis would further clarify the position of Myxophyceae in the plant kingdom (Echlin & Morris, 1965). Perhaps the most interesting aspect of the heterocyst is its function, and this seems to be the main cause of the present curiosity in this cell. Various functions assigned to the heterocyst such as (i) site of filament breakage, (ii) ancient reproductory structure or (iii) a role as an attachment organ can only be incidental functions. Little experimental evidence is available to support the role as site of filament breakage or an attachment organ. T h e proponents of these roles seem to have been impressed by the structural peculiarity of the heterocyst and its situation in algal filaments. Germination of the heterocyst under natural conditions is very rare; the proposal of a role for them similar to akinetes is based on observations in specially constituted culture media. However, a detailed study of heterocyst germination under various conditions is required before forming a conclusive opinion on this controversial subject. Experimental evidence for the inductive role of heterocysts in sporulation in A . cylindrica is quite convincing. This, however, suffers from the weakness of not being valid for all the spore-forming genera. I n such species as A. doliolurn, A. Eaxa and A. afinis Lemm. sporulation begins between the heterocysts and proceeds toward them, whereas in others, for example, A. volxii Lemm. = unispora Gardner, only a single spore is formed on one side of the intercalary heterocysts. Moreover, if the heterocyst induces spore formation, what prevents the spores from differentiating in the very beginning of the life cycle? The most convincing evidence at present implicates the heterocyst in nitrogen fixation. I t is now generally accepted that in heterocystous algae the process of nitrogen fixation is mainly carried out by the heterocysts at least under aerobic conditions. Contrary findings can be grouped under: (i) the use of cultures which have been incubated in anaerobic conditions when nitrogenase activity develops in vegetative cells or (ii) lack of appreciation of the extent to which the nitrogenase activity of heterocysts may vary. Development of nitrogenase activity in vegetative cells in anaerobic conditions indicates the need of a house for nitrogenase protected against oxygen inhibition, and the heterocyst structurally seems suitable for this purpose. Axotobacterlike aerobic nitrogen fixation in Gloeocapsa is, however, interesting and further studies should prove whether this nitrogenase is oxygen insensitive or the alga possesses an efficient mechanism for scavenging oxygen from the active site.


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VIII. SUMMARY

Heterocysts are found in many species of filamentous blue-green algae. They are cells of slightly larger size and with a more thickened wall than the vegetative cells. 2. Structural details of the heterocyst are: the presence of three additional wall layers, the absence of granules, sparse thylakoid network throughout, except at the poles where a dense coiling of membranes occurs. Other characters include the two pores at opposite poles ‘plugged’ with refractive material called the polar granule. 3. Peculiarities in the pigment composition of the heterocyst include an abundance of carotenoids and absence of phycobilins, and a short-wave form of chlorophyll a. 4. Unique glycolipids and an acyl lipid, not found in the vegetative cells of the algae or in other plant cells, are associated with the heterocyst. The glycolipids constitute the laminated layer of the wall and probably regulate diffusion of substances through it, whereas the acyl lipids are supposed to function as carriers and intermediates in the biosynthesis of the wall. 5 . The heterocysts develop from vegetative cells, and the visible changes during differentiation include cell enlargement, synthesis of additional wall layers, disappearance of granules and reorientation and synthesis of the thylakoids. 6. Heterocysts are formed sequentially with characteristic cellular spacing during the growth of cultures in medium free from combined nitrogen. 7. Various sources of combined nitrogen inhibit heterocyst formation when supplied in the culture medium. Ammonium salts are among the most powerful inhibitors. Heterocysts are formed simultaneously and within a short period after transference of ammonia-grown non-heterocystous filaments to ammonia-free medium. 8. Incompletely differentiated heterocysts or proheterocysts are found in cultures grown in the presence of combined nitrogen. If two or more proheterocysts are close together generally a single one develops to maturity after a competitive interaction in medium free from combined nitrogen. This indicates that heterocyst formation is completed in two phases: phase I, synthesis and conservation of macromolecules, which takes place during growth in ammonia-containing medium: and phase 11, morphological differentiation of the heterocyst which is unaccompanied by growth in cell number. In the ammonia-free medium phase 11 quickly succeeds phase I and the whole process appears as a continuum. 9. Heterocyst formation shows a definite requirement for light. Red light favours heterocyst formation, whereas green and blue light do not. T h e effects of light seem to be mainly due to photosynthesis, although some effects may be morphogenetic. 10. Studies with metabolic inhibitors have revealed the involvement of photosynthesis, respiration and protein synthesis in heterocyst formation. Photosynthesis provides carbon skeletons, whereas ATP is most probably supplied by oxidative metabolism. I I . Various functions have been assigned to the heterocyst from time to time. Their role in akinete formation is suggested by (i) the formation of akinetes adjacent to the heterocysts and (ii) prevention of sporulation by detachment of the heterocysts from I.

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the vegetative cells (potential akinetes). Despite substantial evidence for such a role, it is not applicable to all akinete-forming genera. 12. Heterocysts are now widely believed to be the site of nitrogen fixation in bluegreen algae. The main facts in favour of such a role are: (i) fixation of nitrogen by all heterocystous algae, (ii) inhibition of heterocyst formation by combined nitrogen and (iii) direct observations on acetylene reduction by isolated heterocysts. I 3. Some non-heterocystous and unicellular algae, and vegetative cells of heterocystous algae fix nitrogen under microaerophilic conditions suggesting that absence of oxygen favours nitrogenase activity. Heterocysts lack the oxygen-evolving photosystem 11, possess oxidative enzymes, and reduce externally supplied tetrazolium salts - all indicating that they are the most suitable sites for harbouring nitrogenase in aerobic conditions. 14.Heterocysts probably originated in the Precambrian in response to the earth’s changing environment and seem to be the first example of morphological differentiation in the plant kingdom. I thank Professor H. D. Kumar for the loan of reprints during the preparation of this review. LX. REFERENCES

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~

_ I

Pigments and lipids of heterocysts.

Demonstration of C3-photosynthesis in a bluegreen alga, Coccochloris peniocystis.

Changes in thylakoid structure associated with the differentiation of heterocysts in the cyanobacterium, Anabaena cylindrica.

Algae.

Blooming algae.

Plasmodesmata of brown algae.

Marine oscillatoria (Trichodesmium): explanation for aerobic nitrogen fixation without heterocysts.

The occurrence of glutamate synthase in algae.

Lipids of membranes and of the cell envelope in heterocysts of a blue-green alga.

Modifiers of heterocyst repression and spacing and formation of heterocysts without nitrogenase in the cyanobacterium Anabaena variabilis.

P-32 uptake the letic algae.

[Translocation in red algae].

Anticoagulants from marine algae.

Logistic analysis of algae cultivation.

Scale formation in algae.

Photosynthesis and the intracellular inorganic carbon pool in the bluegreen alga Anabaena variabilis: Response to external CO2 concentration.

Taming Toxic Algae Blooms.

Impact of green algae on the measurement of Microcystis aeruginosa populations in lagoon-treated wastewater with an algae online analyser.

Modes of reduction of nitrogen in heterocysts isolated from Anabaena species.

Pathways of assimilation of [13N]N2 and 13NH4+ by cyanobacteria with and without heterocysts.

Hyperspectral imaging of snow algae and green algae from aeroterrestrial habitats.

Research and development for algae-based technologies in Korea: a review of algae biofuel production.

Shewanella algae in acute gastroenteritis.

Parasites in algae mass culture.