Introduction

It is difficult to generalize about the ecological tolerances and distributions along environmental gradients of a group of organisms as large and diverse as the chrysophytes. Many excellent floristic papers lack quantitative ecological data, while others provide ecological data but lack critical microscopical detail. There are many more detailed studies pertaining to the scale-bearing forms than for non-scaled taxa. Thus, we know more about the forms with scales and have much to learn about the non-scaled species. In addition, many methodologies have been used by the different researchers to assemble their data. Despite the apparent obstacles, many generalizations have been made for the chrysophytes as a group as well as for specific and subspecific taxa.

Although there is much scattered information concerning the ecology of the chrysophytes (Kristiansen 1986; Siver & Hamer 1989; Siver 1991), especially for individual taxa, there are few comprehensive studies (Kristiansen 1986; Hartmann & Steinberg 1989; Wee & Gabel 1989). Sandgren (1988) provided an excellent review of the ecology of the group as a whole. Siver (1991) and Eloranta (1989b) summarized ecological tolerances for many taxa of Mallomonas and Dinobryon, respectively; however, similar works are lacking for most other common genera. Kristiansen (1986) and Smol (1986, 1990, this volume) provide excellent starting points for the use of the chrysophytes as biological indicators and in paleolimnological work, respectively.

Introduction

Over 200 non-protein amino acids occur naturally in plants (Rosenthal, 1982). With a few exceptions, these amino acids exist in unconjugated forms and many are associated with toxic properties (D'Mello, 1991). Legumes contain higher concentrations and a more diverse range of non-protein amino acids than any other plant species, and the seed is generally the most concentrated source of these substances.

The toxic non-protein amino acids are a distinguishing feature of many tropical legumes (D'Mello, 1992), contributing significantly to the noxious effects of a number of grain and forage legumes, including Canavalia ensiformis, Indigofera spicata, Leucaena leucocephala and at least three Lathyrus species. However, there is evidence of considerable variation in the concentrations of non-protein amino acids in different species of the same genus (Aylward et al, 1987). Toxic non-protein amino acids also occur in non-leguminous plants, both tropical (e.g. Blighia sapida; Cycas circinalis) and temperate (e.g. Brassica species).

The toxicity of non-protein amino acids has been observed in insects, laboratory and farm animals and in humans, but there are striking differences among animal species in their sensitivity to these compounds. Factors such as diet, duration of feeding and geographical differences in microbial ecology in ruminants may also modulate the response to these amino acids.

The role of the non-protein amino acids in plants remains a matter of some debate. However, there is mounting evidence that these compounds form part of the chemical defence system against predation and disease (Rosenthal & Bell, 1979).

Introduction

Ethylene is the plant growth regulator that controls ripening and senescence in plants. It is produced from methionine via the formation of S-adenosyl-l-methionine, which, in turn, forms the non-protein amino acid, 1-aminocyclopropane-1-carboxylic acid (ACC) (Fig. 1). When ACC was first recognized as the immediate substrate for ethylene formation, it became apparent that all kinds of plant tissues had an active and constitutive enzyme capable of catalysing the conversion of ACC to ethylene, as high rates of ethylene production were observed when plant tissues were supplied with ACC (Cameron et al., 1979). The rate of ethylene production when tissues were fed ACC was always far greater than when they were fed methionine under the same conditions (Yang & Hoffman, 1984). During climacteric fruit ripening and flower senescence an initially low ACC oxidase activity increased in line with the rise of ACC synthesis and ethylene production (Yang & Hoffman, 1984). However, despite the substantial ACC oxidase activity that was readily observed with plant tissues, in vitro activity could not be detected. When tissues were homogenized, methionine was readily converted enzymatically to ACC, but there was no ethylene formation, and ACC supplied to tissue homogenates was not converted to ethylene. These observations led to the notion that ethylene formation required membrane integrity (Yang & Hoffman, 1984). This view was supported by the sensitivity of ACC oxidase to osmotic shock, detergents and protonophores, and by the discontinuities in the Arrhenius plots for ethylene production (see Yang & Hoffman, 1984; Kende, 1993).

Research activity involving algae in the algal classes Chrysophyceae and Synurophyceae (collectively, ‘chrysophytes’) has increased dramatically over the last decade. Chrysophyte algae are primarily freshwater flagellate organisms that typically constitute a dominant or subdominant portion of the phytoplankton biomass in lakes with moderate to low productivity. They have long been under-studied by aquatic ecologists because of difficulties in culturing and preserving chrysophytes, and because of the tendency to study eutrophic, human-impacted lake systems in which chrysophytes are relatively rare. However, the critical importance of chrysophytes in carbon flux through oligotrophic, freshwater food webs has been the subject of considerable recent research. The mixotrophic nutritional capacity of most chrysophytes provides them with a distinct advantage in nutrient-poor lakes. Chrysophyte-rich lakes are often poorly buffered and are thus sensitive to anthropogenic acidification. Because of the extreme interest in lake acidification in both North America and Europe, siliceous chrysophyte microfossils (resting cysts, cell scale layers) have rapidly become primary paleolimnological research tools. Chrysophyte microfossil research has, in turn, stimulated considerable renewed interest in their biogeography and in environmental factors influencing seasonal and spatial distribution patterns. This activity has revealed fundamental gaps in our knowledge of chrysophyte nutritional physiology and basic metabolism. Chrysophyte algae also continue to occupy a central position with regard to evolutionary relationships among groups of chromophyte, chlorophyll a and c containing algae (diatoms, chrysophytes, brown algae, dinoflagellates, etc.) and also in linking chromophytes to several non-photosynthetic protistan groups (amoebae, heliozoans, choanoflagellates, oomycete molds). These relationships have been the subject of considerable ultrastructural phylogenetic research over the last 15 years, and chrysophytes continue to be pivotal in current molecular phylogenetic studies of photosynthetic protists.

Glycine decarboxylation and photorespiratory metabolism in C3 plants

In higher plants which carry out C3 photosynthesis, photosynthetic and photorespiratory metabolism is based on the action of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase and the regeneration of its substrate ribulose-1, 5-bisphosphate by the reductive pentose phosphate pathway (RPP) or Calvin cycle. These reactions occur in the chloroplast. The product of CO2 fixation by RuBP carboxylase is two molecules of 3-phosphoglycerate (a three-carbon compound; hence C3 photosynthesis) which is either exported from the chloroplast as triose phosphate for sucrose synthesis in the cytosol or metabolized to form starch within the chloroplast or used for regeneration of RuBP. Oxygen competes with CO2 for the active site of RuBP carboxylase/oxygenase leading to an oxygenase reaction which reduces the rate of CO2 assimilation. The products of the oxygenase reaction are 3-phosphogly cerate and phosphoglycolate (a two-carbon compound). The production of phosphoglycolate represents a drain of carbon away from the RPP and to recover this carbon the phosphoglycolate is metabolized through a series of reactions involving enzymes in the chloroplasts, peroxisomes, and mitochondria. In the course of this pathway two molecules of glycine (i.e. four carbon atoms) are metabolized to one molecule of serine, CO2 and NH3. Serine is metabolized further to 3-phosphoglycerate and so three out of four carbon atoms entering the pathway are returned to the RPP (Husic, Husic & Tolbert, 1987; Ogren, 1984). The light energy requirement of photosynthesis and photorespiration is for the synthesis of ATP and NADPH via the electron transport pathway of the chloroplast.

This account covers a period of about 200 years – from the early beginnings of light microscopy until electron microscopy became established in the middle of the twentieth century. It takes its beginning in the middle of the eighteenth century, in rural Denmark at idyllic Frederiksdal, a manor house 20 km north of Copenhagen. In 1750, Countess Catharina Schulin had lost her husband Count Johan Siegesmund Schulin, who had held a high position in the Danish government administration. She was left alone with two children, a large estate, and an enormous staff of servants. She sought comfort in religion and summoned young theology students from the University to preach for her (Anker 1943).

Among these was 20-year-old Otto Friedrich Miiller (Fig. 1.1). He won the friendship of the countess, and after 3 years he was appointed as a private tutor to her young son. He took this position very seriously, so that the young count at the age of 10 years and 9 months was able to pass the entrance examination to the University. Later, Miiller accompanied the young count on the European tour that was traditional for young noblemen.

Müller had leisure time during his appointment and the beautiful surroundings of Frederiksdal stimulated his interest in natural history. Countess Schulin was interested not only in religion, but also in many aspects of culture and science. She was extremely pleased with Müller's service and encouraged and supported him in his scientific studies.

Introduction

Chrysophyte algae: ecology and biogeographic distribution

The great majority of planktonic chrysophytes (algal class Chrysophyceae sensu Hibberd 1976; incl. Synurophyceae, sensu Andersen 1987) are rather delicate, golden-colored flagellates. Both unicellular and colonial chrysomonads are common in lake plankton and they exhibit three distinct types of cell coverings: ‘naked’ cells (cell membrane only), cells in expanded organic loricas, and cells covered with ornamented siliceous scales and/or bristles. This morphological diversity may affect their palatability for herbivores or may increase the effective diameter of chrysophyte cells as zooplankton ‘food particles’. Chrysophytes range in natural particle size from a few micrometers to several hundred micrometers in diameter; larger colonies are mostly spherical (Synura, JJroglena, Chrysosphaerella), but some are dendroid (Dinobryon) or linear (Chrysidiastrum). Chrysophyte algae demonstrate seasonally restricted population cycles in lakes (Sandgren 1988); they produce siliceous resting cysts and probably recruit annually from sedimentary ‘seed’ populations of these cysts (Sandgren 1991).

Chrysophytes are among the most poorly studied freshwater phytoplankton with regard to their nutrition, physiology and ecology. Those genera of interest here are phototrophs, but many also have a facultative or obligate capacity for supplementary phagotrophic and osmotrophic feeding (Sanders 1991; reviewed in Sandgren 1988; also see Holen & Boraas, this volume). Chrysophyte algae are frequently biomass dominants, together with other algal flagellates, in the myriad of small, softwater, and largely oligotrophic lakes of the north-temperate regions of North America and Scandinavia (as summarized in Sandgren 1988).

The previous chapter has described the roles that glycine decarboxylase (GDC) and serine hydroxymethyltransferase (SHMT) play in converting photorespiratory-derived glycine to serine, primarily to recover some of the carbon from the glycolate produced by the oxidative reactions of RUBISCO in C3 plants. As well as being needed for protein synthesis, glycine and serine are precursors of a variety of molecules essential to the growth and development of plant tissues. Glycine is required for the synthesis of, amongst other things, glutathione, porphyrins such as leghemoglobin in nitrogen-fixing root nodules, and purines which are needed for nucleic acid synthesis and, in some nodules, for ureide production. Serine plays a similar role as a precursor of biomolecules, including phospholipids, tryptophan and cysteine, and, under some stress conditions is involved in the synthesis of glycinebetaine (see chapter by Gorham, this volume, and Rhodes & Hanson, 1993). The interconversion of glycine and serine, with the concomitant production of methylene tetrahydrofolate, is also important to the plant as a source of one carbon units (see Cossins, 1980). In C3 leaves, photorespiration provides substantial amounts of glycine and serine which can be used for such syntheses. In non-photosynthetic tissue such as roots and developing and germinating seeds, there is a need for glycine and serine just as in the leaves, but there is no photorespiration to produce high levels of glycine and serine. Alternative, nonphotorespiratory routes for the production of glycine and serine exist in such tissues, as well as in the leaves of C3 plants.

Previous concepts of classification

Until recently, most concepts of higher taxa in the Chrysophyceae have been based on some combination of vegetative morphological features and characteristics of the motile cells (such as flagellar number and position). For a survey on the various systematic treatments of the Chrysophyceae by earlier workers (Pascher 1914; Fritsch 1935; Bourrelly 1968; Fott 1971; Bold & Wynne 1978; Christensen 1980; Ettl 1980; and Kristiansen 1982) the reader is referred to Round (1986). In the present account (see Tables 3.1–3.3) special reference is given only to the more recent treatments by Starmach (1985) and Kristiansen (1986, 1990).

Within the class Chrysophyceae, Starmach (1985) distinguished three subclasses: the Heterochrysophycidae, the Acontochrysophycidae and the Craspedomonadophycidae. The Craspedomonadophycidae of Starmach included the choanoflagellates (families Monosigaceae and Salpingoecaceae) and the genus Phalansterium (family Phalansteriaceae) which are now known from electron microscopic studies to have no structural similarities with any group of algae (Hibberd 1986), and it is now generally accepted that they should be classified only as Protozoa. Starmach's scheme more or less followed that of Bourrelly (1968, 1981), in which three different lineages were recognized: the uniflagellate order Chromulinales in the subclass Heterochrysophycidae, the biflagellate order Ochromonadales in the subclass Heterochrysophycidae, and several aflagellate orders in the subclass Acontochrysophycidae.

Introduction

The diversity and morphology of scales and scale-like structures (e.g., spines, spine-scales, bristles) are remarkable among the different protistan groups, and the mechanism of their assembly and deployment can vary considerably (for review, see Romanovicz 1981). Arguably the most spectacular scale-bearing algae are found in the division Chrysophyta, which includes the organisms under investigation here: Mallomonas splendens (G.S. West) Playfair em. Croome, Dürrschmidt & Tyler (Synurophyceae) and Apedinella radians (Lohmann) Campbell (Pedinellophyceae). These two species collectively exhibit a wide range of surface features, some of them unique, and are excellent experimental systems for studying the development of scales and scale cases.

A number of cytological techniques have been used to investigate scale formation and development, most notably scanning and transmission electron microscopy. The formation of synurophycean scales (including the scale-like ‘bristles’ of Mallomonas) has been followed in several species at the ultrastructural level (e.g., Mignot & Brugerolle 1982; Brugerolle & Bricheux 1984). The exact manner of scale deployment onto the surface, however, is unknown, although two possible mechanisms have been put forward (Leadbeater 1990; Siver & Glew 1990). Recently, in vivo observations of bristle secretion and deployment in M. splendens have been made using image-enhanced video microscopy, and corroborated ultrastructurally with thin-sectioned material (Beech et al. 1990).

The development of immunocytochemical techniques has extended our knowledge of the cytoskeletal components active in these processes as well as the nature and role of surface molecules associated with the scale layer.

Background

Between 75% and 85% of the mature wheat grain is starch, so above all else, yield is a measure of the whole-plant processes that culminate in starch deposition in the grain. Protein percentage, on the other hand, is a ratio value, and whilst not independent of yield is obviously an expression of nitrogen metabolism. The rates and durations of both starch and protein deposition in the endosperm of wheat all appear to be independent events controlled by separate mechanisms (Jenner, Ugalde & Aspinall, 1991). It is this independence that gives the opportunity to manipulate specific responses in the plant that culminate in starch and protein deposition, whether the attempts at improvement be genetic or agronomic.

The relationship between substrate supply and dry matter deposition is different for starch and protein, and the responses change during grain development (Jenner et al, 1991). During the grain filling stage (10–15 days after anthesis until the onset of maturity), the rate of starch deposition in healthy plants is mainly influenced by sink-limited factors, that is by factors that operate within or close to the grain itself. By contrast, deposition of protein is influenced to a much greater extent by source-limited factors, that is by factors of supply. Increasing amino acid supply to developing grains leads directly to increases in protein deposition. Within this context, however, the levels of substrate within the endosperm (sucrose and amino acids respectively) appear inconsistent with what may be expected.

The branched chain amino acid biosynthetic pathway has received considerable attention in recent years because different chemical classes of highly successful commercial herbicides kill plants by inhibiting this pathway. This discovery has led to identification and design of other inhibitors of this pathway that are also herbicidal. Our recent studies on the mode of action of imidazolinone herbicides have provided clues that may help clarify the role of 2-ketobutyrate and 2-aminobutyrate in the phytotoxic effects of these herbicides. These studies have also provided insight into understanding the regulation of carbon flow through the branched chain amino acid biosynthetic pathway in plants. We have also identified a new form of threonine dehydratase (TD; EC 4.2.1.16) that may have a crucial function in nitrogen metabolism in senescing leaves.

Accumulation of 2-KB/2-AB vs phytotoxidty of AHAS inhibitors

The imidazolinone and sulfonylurea families of highly successful commercial herbicides kill plants by inhibiting acetohydroxyacid synthase (AHAS; EC 4.1.3.18), the first common enzyme in the pathways leading to the biosynthesis of valine, leucine and isoleucine. These extremely potent herbicides kill plants at application rates of grams per hectare. The high potency of AHAS inhibiting herbicides is of great interest because inhibitors of other enzymes in the branched chain amino acid pathway require much higher rates to kill plants (Wittenbach, Aulabaugh & Schloss, 1991; Shaner & Singh, 1992).

Introduction

Excessive accumulations of algae in freshwater lakes and coastal marine environments have been observed for centuries. Homer's Illiad mentions discoloration of the sea, and the Bible contains a reference to ‘the bloodied waters of the Nile’. Charles Darwin apparently observed a ‘red tide’ off the coast of Chile, and North American Indians would not eat shellfish from ‘shining waters’ based on previous experiences with algal-bloomrelated shellfish poisonings (Red Tide Newsletter 3(2), April 1990). Among the causes most often cited are enrichment of aquatic systems with nutrients from human activities (Vallentyne 1974; Smayda & White 1990). While there is strong evidence that excessive supply of nitrogen and phosphorus and other nutrients is often the underlying cause of algal blooms, it is also clear that bloom development depends upon the coming together, in appropriate combination, of a number of critical biotic, physical and chemical environmental factors (Paerl 1988).

The implication of the early historical evidence of algal blooms is that the consequences of accelerated human population growth and contemporary urbanization and industrialization activities cannot be the only causative factors for algal blooms. For example, there were especially intense blooms of the dinoflagellate Alexandrium cantenella off the Norwegian coast in 1988, which may have been in response to abnormally high water temperatures. Paleo-oceanographic evidence (B. Dale, University of Oslo, unpublished data) suggests that similar blooms developed as far back as 2000 years ago when human-induced influences were undoubtedly negligible.