Introduction

Cyanogenic glucosides are secondary plant products which, upon hydrolysis, release HCN. Cyanogenic glucosides are found in more than 2000 plant species, including the agriculturally important sorghum and cassava. Insufficient removal of the cyanogenic glucosides present in the cassava tubers constitutes a potential health hazard for the millions of people who are dependent on these tubers as their staple food. The presence of cyanogenic glucosides in cassava tubers have been shown to improve their resistance against the cassava root borer (Bellotti & Arias, 1993). However, in other cyanogenic plants, the cyanide released may be more harmful to the host plant than to the pest organism. This is the case in the cyanogenic rubber tree (Hevea brasiliensis), where elevated amounts of cyanogenic glucosides result in increased sensitivity to attack by the fungus Microcyclus ulei (Lieberei, 1986). Similarly, the presence of the cyanogenic glucoside epi-heterodendrin in the epidermal cells of barley seedlings is correlated with an increased sensitivity to the mildew fungus Erysiphe graminis Pourmohseni & Ibenthal, 1991. In these cases, the HCN released upon infection is impairing the plant defence responses, either by inhibiting the synthesis of phytoalexins or by inhibiting polyphenol oxidases (Lieberei et al. 1989). The presence of even minor amounts of epi-heterodendrin in barley malt causes a problem in the brewing industry (Cook et al. 1990). During the distilling process cyanide is liberated, which results in the formation of the carcinogenic ethylcarbamate, formed from cyanide and ethanol.

Introduction

Chrysophytes have long been recognized to be powerful indicators of environmental conditions (e.g. Siver, this volume); however, only recently have they been used extensively in paleoecological studies. In many ways, the increased use of chrysophyte microfossils has closely tracked the heightened interest and application of paleoecological approaches to the study of environmental change (see Davis 1989; Smol 1990a, b, 1992; Battarbee 1991; Smol & Glew 1992; and Charles et al. 1994 for recent reviews and commentaries). Historical perspectives, such as those that can be gleaned from lake sediments using paleoecological approaches, have now been melded into a wide array of studies that cover both theoretical and applied aspects of limnology (Smol 1990a). With these proxy data, long-term environmental conditions and variability can be assessed, hypotheses can be generated and tested, and models can be verified.

This chapter highlights some of the recent advances in the use of chrysophytes as paleolimnological markers, and stresses examples that I believe are most relevant to phycologists. Because of the increased volume of literature, it is no longer possible to provide a thorough synthesis in an article of this size. The reader is referred to reviews by Adam & Mahood (1981), Cronberg (1986a, b Kristiansen (1986) and Smol (1987, 1988a), who summarize much of the historical literature on chrysophyte microfossils and provide a point of departure for this chapter. Smol (1990c) compiled a bibliography of most work on chrysophyte-based paleolimnology published in the 1980s.

This book arose from a meeting at Rothamsted Experimental Station in September 1993, sponsored by the Plant Metabolism Group of the SEB. It was one of several meetings, on a wide variety of topics, which helped to celebrate the 150th anniversary of Rothamsted, which was founded by John Bennett Lawes in 1843. At that time, Lawes was involved in the commercial production of superphosphate fertilizer, and the experiments begun by Lawes and his co-worker Gilbert investigated many aspects of plant nutrition. That the conference (and this book) dealt with amino acids, end products of nitrogen assimilation in plants, is most appropriate considering the pioneering work of Lawes and Gilbert on plant nitrogen nutrition.

Amino acid biochemistry in plants has been a major topic of research at Rothamsted for more than 20 years, and many major advances have been made: the discovery of the glutamate synthase cycle for the assimilation of ammonia, the first description of the photorespiratory nitrogen cycle, and detailed genetic, biochemical and molecular analysis of these processes; the biochemistry and genetics of amino acid biosynthesis, particularly aspartate-derived amino acids; and most recently the biochemistry of amino acid-derived secondary metabolites. This work has involved many other researchers from laboratories all over the world, and it was a great pleasure to welcome so many past and present collaborators to the meeting, and to read of their latest work in the chapters of these proceedings.

Aliphatic glucosinolates are sulphonated thioglucosides which are found in all hitherto investigated plants of the order Capparales, which includes the genera Brassica and Arabidopsis. Although these secondary metabolites share the general structure (see previous chapter and Fig. 1), differences in their ‘R’ substituents enable classification into several distinct structural classes. The aliphatic glucosinolates found in Brassica typically include methylthioalkyl, methylsulphinylalkyl, alkenyl and hydroxyalkenyl homologues of propyl, butyl and pentyl glucosinolates. A.thaliana contains a similar variety of glucosinolates, with the addition of hydroxyalkyl glucosinolates and trace levels of longer side-chain homologues (Hogge et al, 1988). Following disruption of cell integrity, aliphatic glucosinolates are hydrolysed by endogenous β-thioglucoside glucohydrolases (‘myrosinases’; EC 3.2.3.1) to give a complex mixture of products, of which D-glucose, sulphate and isothiocyanates are major components (Cole, 1976; Benn, 1977; Fenwick, Heaney & Mullin, 1983). In addition to being responsible for the characteristic flavour of cruciferous condiments, hydrolytic products of glucosinolates such as isothiocyanates, thiocyanates and nitriles are known to impart antinutritional properties to protein-rich oilseed ‘meals’ commonly used as animal-feed (Bell, Benjamin & Giovanetti, 1972; Fenwick, 1984). In particular, isothiocyanate derivatives of hydroxyalkenyl glucosinolates undergo spontaneous cyclization to produce substituted oxazolidine-2-thiones (Macleod & Rossiter, 1987) which have potent goitrogenic properties (Astwood, Greer & Ettlinger, 1949; Langer, 1966). The occurrence of these antinutritional compounds in seed-meals of major oilseed Brassica crops has led to efforts to reduce the level of aliphatic glucosinolates in the seeds of oilseed rape.

Introduction

Fertilisation in the brown alga Fucus involves species-specific interactions between biflagellate sperm and spherical eggs (Bolwell et al., 1977; Evans, Callow & Callow, 1982; Callow, Callow & Evans, 1985; Callow, Stafford & Green, 1992). We are interested in two related aspects of Fucus gamete cell surfaces: 1) How are the cell surface molecules organised? and 2) What is the molecular basis of recognition and the associated cell responses that occur within a few seconds or minutes of gamete fusion? Such studies in higher plants are difficult because the gametes are embedded within tissues, and plasma membrane based receptors have limited accessibility because of intervening cell walls. In addition, it is still relatively difficult to obtain gametes in sufficient numbers from higher plants compared with Fucus from which naked gametes are released in large enough quantities to allow detailed biochemical studies (Bolwell, Callow & Evans, 1980; Stafford, Callow & Green, 1992a). Thus the Fucus system has much to offer, and hopefully the findings will be relevant to gamete interactions in higher plants. This review will focus on how we have used a combination of biochemical and immunological approaches to study: 1) the organisation of the Fucus egg cell surface and 2) the role of sperm proteins in egg binding and the triggering of cell wall release.

Summary

The highly specialised angiosperm male gametophyte is both the site of production and the vehicle by which the male gametes are transported to the embryo sac to participate in fertilisation. Because of its relative simplicity compared with the sporophyte and accessibility for cytological and molecular analysis, the male gametophyte represents an excellent system in which to unravel the molecular basis of gene regulation and cellular differentiation in plants. The intent of this chapter is to review evidence for haploid gene expression in the developing male gametophyte, emphasising in particular the contribution of molecular cloning and transgenic approaches. cDNA cloning and RNA analysis has led to the characterisation of more than 30 microspore or pollen-expressed genes from 12 different plant species which are preferentially activated at particular stages of development. Two broadly defined groups of pollen genes are recognised; the ‘early genes’, which are activated prior to pollen mitosis I (PMI), and the ‘late genes’ which are first activated after PMI. Promoter studies have demonstrated differential gene expression between the vegetative and generative cells and insight into the significance of PMI for late gene activation. Detailed analyses of the promoters of several late pollen genes have led to the identification of several distinct cis-regulatory elements which control both the level and specificity of haploid gene expression. Some of these regulatory elements are functionally conserved among homologous promoters from diverse plant species and among different promoters within a single species.

Introduction

Daylength is the most powerful environment factor in the regulation of the onset of flowering (Vince-Prue, 1975). Changes in photoperiod through the year provide an unambiguous index of seasonal progression. Through the ability to detect and respond to daylength, plants can tailor flowering to seasonal changes in climate, and can also ensure synchrony of flowering to facilitate outbreeding. Plants are classified as short-day plants (SDP), long-day plants (LDP), day neutral plants (DNP) or combinations of these. SDP flower in response to days shorter than a critical daylength, LDP flower in response to days longer than a critical daylength and DNP flower irrespective of daylength. Some plants show a requirement for particular sequences of daylengths or other combinations of the above basic types (Thomas & Vince-Prue, 1984). Photoperiodic requirements may themselves be the only determining factors, but frequently they are overlaid on other developmental or environmental factors, the most important being juvenility and vernalisation (Thomas, 1993).

Photoperiodic mechanisms

Daylength is generally accepted as being perceived by the leaves in both SDP and LDP, rather than in the apex where the transition to flowering occurs. When a permissive daylength is perceived, a semi-stable change in the properties of the leaves occurs. This can be demonstrated by the ability of such leaves to cause flowering when grafted to plants maintained in non-permissive daylengths (for example, Zeevaart, 1969). The change in the leaf is called induction and the molecular basis of the change is unknown.

Introduction

It is a truism of biology that most of the key processes typifying the living state in eukaryotic cells involve intracellular movement. Growth, morphogenesis, division, secretion, interaction with other cells, all require continuous physical redistribution of cell components, whether of organelles, membranes, vesicles, nuclei or chromosomes: in this respect the motility systems of the cell may be said to lie near to the heart of eukaryotic physiology. Our purpose in this outline review is to summarise some features of the life of the male gametophyte generation of the flowering plants as seen from this point of view.

Overt vectorial movement in the pollen tube itself is expressed in three main ways: in the circulation along its length of organelles and other cytoplasmic inclusions; in the net movement away from the parent pollen grain of the vegetative nucleus, the generative cell and the sperms formed from it; and in the transport and release through the plasmalemma of secretory vesicles concerned with the insertion of precursor materials into the growing wall and the release of enzymes into the environment. All of these processes are conducted within the confines of a single tip-growing, partly heterotrophic, cylindrical cell.

It is well established for both plant and animal cells that intracellular motility depends on interaction with cytoskeletal elements. Two systems are positively known from the pollen tube: one micro tubule based, and the other actin-fibril based.

The articles in this volume provide a stimulating overview of several aspects of current research in plant reproductive biology. Some convincingly illustrate how age-old problems of plant reproductive systems are proving to be accessible to the awesomely powerful methods of molecular biology, while others show how this approach can complement – and be complemented by – other new techniques for investigating intracellular processes and cellular interactions at levels beyond that of the gene. In the light of these many current developments, it is surely not overly optimistic to suggest that we are entering a new era in the exploration of plant reproductive phenomena.

So, in what directions might research be expected to progress now? While defining priorities is always fraught, it is at least permissible to attempt to identify a few parts of this very wide subject where there is a real promise that some of the outstanding problems could yield to the application of the new methodologies. In this connection it is important for the plant scientist to keep an eye on what is happening in animal and microbial cell biology; after all it is from these much more fully populated and much better resourced fields that we borrow most of our technology. We need also to bear in mind that sexual reproduction in plants involves essentially all the basic processes of development: cell nutrition, division, growth and communication, tissue differentiation and organ morphogenesis.

Systems for inter-organ communication

In order to successfully survive, all living organisms require a system to respond to the continuously changing environment, and to regulate the various developmental processes in cells and organs at different locations in the body. The following discussion highlights the communication systems in plants with special emphasis on reproductive aspects.

In animals, two major systems are involved in inter-organ communication, i.e. the nervous system and the hormone system. In addition, the immune system uses different classes of chemical messengers (e.g. interleukins, interferons, nitric oxide) to coordinate the defence response (Norman & Litwack, 1987; Roitt, Brostaff & Male, 1993). Although the metabolism of individual plant and animal cells may show many similarities, there is no a priori reason why the mechanisms, involved in the responses to environmental cues and in communication between different organs should in any way be the same. However, systems showing some similarities to the animal nervous system have been found in plants. For instance, mechanisms have evolved to respond within seconds to mechanical perturbation in Drosera (sundew) and Mimosa pudica. The movements of the leaves of Mimosa pudica are regulated by motor organs (pulvini) and are the result of turgor variations in the cortical (motor) cells of these organs. It has been shown that ionic migration, in particular of K+ and C1−, appears in the pulvinus, concomitant with the turgor changes (Satter & Galston, 1981; Fleurat-Lessard et al., 1988).

Introduction

Senescence is a process usually considered to encompass those events which lead irreversibly to death. However, whilst the term ‘flower senescence’ is frequently encountered, it rarely relates to the flower as a whole but more particularly to those parts of the flower which are regarded as attractive, that is the perianth parts. In fact, as some parts of the flower senesce, other floral organs are still developing. For example, in species exhibiting protrandy, the anthers may well senesce as the stigma expands and becomes receptive to pollination. A further contrast is to be found when petal and leaf senescence are compared; the degeneration of the chloroplast would normally be regarded as senescence in leaves, however, in petals such a process occurs early on and is considered part of the process of petal development.

The process of flower senescence differs greatly from species to species; in some the petals wilt and may eventually abscise, in others abscission of the perianth occurs whilst fully turgid; in yet others a change in colour of all, part, of the perianth portends the ageing process. From this it is clear that both the physiology and structure of the petals undergo changes during senescence. This review endeavours to describe some of the physiological and structural changes that occur in these varied strategies and the factors, particularly pollination, which may initiate the senescence of the perianth parts.

Introduction

Cauliflower (Brassica oleracea L. var. botrytis) is a member of the genus Brassica in the family Brassicaceae. It is a major economic crop which is marketed as a large, white, pre-floral, compact curd. The cauliflower curd is, however, susceptible to a number of morphological defects which can seriously reduce its commercial value (King, 1990). The genetic determinants that control floral meristem development and flower morphogenesis in cauliflower have not been approached at the molecular level until recently. Medford, Elmer and Klee (1991) have now isolated and partially characterised a number of meristematic genes expressed in the immature cauliflower curd. In addition, Anthony, James and Jordan (1993) have isolated a homologue of the Antirrhinum flo gene (Coen et al., 1990) that is thought to be involved in regulating floral initiation.

In this review, we will briefly describe the morphology and physiology of the cauliflower, with particular emphasis on floral development. In addition, we will describe the isolation of genes involved in floral initiation and discuss how these genes may interact to control cauliflower morphology.

Morphology and physiology of cauliflower floral development

Five stages of development have been recognised between vegetative growth and flowering in cauliflower (Margara & David, 1978): 1. The vegetative stage in which the small pointed shoot apex is surrounded by leaf primordia. These arise acropetally in a spiral succession and axillary branches do not develop; 2. Initiation of inflorescence results in precocious formation of axillary buds at the apex to form clusters of meristems and bracts; 3.

Introduction

Mutants affecting sexual development in plants are relatively rare. Most of them concern cytoplasmic or nuclear male sterility, while a series of meiotic mutants in maize are well characterised (Kaul & Murthy, 1985). Other mutants, of the sex conversion type, have been described in maize and cucumber, two monoecious plants (Irish & Nelson, 1989; Malepszy & Niemerowicz-Szczytt, 1991).

Among flower pattern mutants, those affecting floral organs in whorls 2 and 3 exhibit various degrees of homeotic transformation of stamens into carpels e.g. apetala3 in Arabidopsis thaliana and deficiens in snapdragon (Jack, Brockman & Meyerowitz, 1992; Schwarz-Sommer et al., 1990). The corresponding genes have been cloned and represent transcription factors belonging to the MADS class.

We have recently described two asexual mutants in the dieocious Melandrium album, following irradiation of pollen with low doses of gammarays (Veuskens et al., 1992). Briefly, the mutants 5K63 and 8K40 have normal perianth organs (sepals and petals), while lacking both male and female reproductive organs: vestigial stamens and a ‘finger-like projection’ instead of carpels are present in whorls 3 and 4 respectively. Both mutants have a deletion covering respectively 12 and 21% of the Y-chromosome.

Here we report on the use of these asexual/Y deletion mutants in cDNA subtraction cloning and exploit them in refining the current understanding of sex determination in a plant X/Y system

Angiosperm pollen represents the pinnacle of an evolutionary progression towards gametophyte miniaturisation that probably began with the development of a toughened wall surrounding the zygote of charphycean algae, the progenitor of the land plants, some 400 million years ago (Delwiche, Graham & Thomson, 1989). The combination of acute vulnerability and central importance in the life-cycle of sexually reproducing plants inherent in the usually brief life-cycle of the male spore has driven the assembly of various adaptive features that endow its protective capsule with remarkable properties. Foremost among these is the unparalleled combination of physical strength, chemical inertness and resistance to biological attack of the outer wall, or exine, due principally to its major structural component, sporopollenin. The evolutionary history of sporopollenin, and that of the spores of land plants, is indivisible (see Chaloner, 1976). Fossil green algae dating back to the Devonian period have been shown to contain sporopollenin (Wall, 1962) and there are reports that sporopollenin also occurs in fungi (see Shaw, 1971) indicating an origin predating the appearance of plants. In an article that fuelled the debate about the existence of extra-terrestrial life, Brooks and Shaw (1969) also reported the presence of sporopollenin in meteorites and suggested that the origin of sporopollenin could even predate life on earth.

However, the exine is often much more than a protective shield. In angiosperms, for example, the pollen wall of many species has become modified to carry specific self-incompatibility proteins that promote outbreeding.