The vacuole is, in terms of volume, the dominant organelle of the vast majority of mature plant cells. Many of its functions have long been recognised (Dainty, 1968; Boiler & Wiemken, 1986; Matile, 1987; Raven, 1987). Thus, the vacuole provides a storage compartment for nutrients and metabolites, exhibits lysosomal characteristics as a major lytic compartment, and (by virtue of its size and relative metabolic inertness) enables the cell to achieve a large volume without compromising the high cytoplasmic surface-to-volume ratio that is necessary for efficient gas and nutrient exchange with the environment.

Increasingly, however, additional functions for the vacuole are being discovered. The presence of ion translocating phosphohydrolases at the vacuolar membrane (the tohoplast) implies that the vacuole plays an important role in cellular energetics (Rea & Sanders, 1987). Indeed the H+-pumping pyrophosphatase at this membrane appears (in photosynthetic tissue at least) to constitute the only means of hydrolysis of cytosolic pyrophosphate (PPi), which suggests that the organelle must be primarily responsible for the removal of PPi produced during biosynthetic reactions (Weiner et al, 1987). Furthermore, in accord with its role as a storage organelle, the vacuole seems likely to constitute a major source of second messengers which can be released into the cytoplasm during signal transduction (Boudet & Ranjeva, 1989).

This chapter addresses some of the interactions that occur between primary nitrogen assimilation and photosynthetic and respiratory metabolism. Much of the pioneering work in this area was carried out by Syrett (Syrett, 1953, 1956a,b, 1981) and Bassham, Kanazawa and co-workers (Bassham et al, 1981; Kanazawa et al, 1970, 1972, 1983). It is not coincidental that this work has often employed algal cells. Single-celled algae have C/N ratios between 7 and 12 compared with >20 in higher plants. Therefore, the relative importance of nitrogen in carbon metabolism of single-celled algae is much greater than in higher plants and the interactions between N assimilation and carbon metabolism are much more apparent.

For several years our group has studied the interactions between photosynthesis, respiration and N assimilation in the green alga Selenastrum minutum. Our approach has been to grow this alga in chemostat cultures under N-limited conditions. The addition of a source of inorganic nitrogen (NH4+, NO2- or NO3-) to these cells activates the assimilation of N into amino acids allowing study of the corresponding changes in metabolism. This chapter reviews some of the progress made using this system.

Nitrogen assimilation by N-limited and N-sufficient algal cells

The primary assimilation of inorganic N into amino acids and protein requires ATP, reducing power and carbon skeletons in the form of ketoacids. In photosynthetic tissues, ATP and reducing power are supplied by either photosynthetic or respiratory processes; however, most of the carbon skeletons used in amino acid synthesis are intermediates of respiratory metabolism (Fig. 1).

Crassulacean acid metabolism (CAM) represents the third major subdivision of photosynthetic carbon assimilation types in green plants alongside the C3 and C4 pathways. As with the C4 pathway, the CAM pathway can be regarded as an ancillary biochemical mechanism, serving to provide CO2 at elevated concentrations for fixation in the Calvin cycle of C3 photosynthesis. In contrast to the C4 pathway, however, all the key biochemical components of carbon assimilation in CAM plants are to be found within individual mesophyll cells. CAM, in essence, is a cellular phenomenon, and this highlights the importance of regulating carbon flow between organelles at the subcellular level. The comparative biochemistry of CAM plants has been the subject of excellent reviews (Kluge & Ting, 1978; Osmond, 1978; Osmond & Holtum, 1981; Winter, 1985; Luttge, 1987; Griffiths, 1988; Leegood & Osmond, 1990). These sources can be consulted for detailed information on metabolic pathways and enzymic characteristics. We shall focus here on the significance of subcellular compartmentation for the control of carbon flow in the CAM pathway. While describing the metabolic interconversions characteristic of each compartment, our particular aim is to show how the regulation of metabolite flux between organelles is also fundamental to the carbon assimilation process in CAM plants. Our understanding of these transport processes is improving, but we are only just starting to unravel details of their molecular mechanisms.

Carnitine is widely distributed in the tissues of animals, plants and microorganisms. However, whilst there is an abundance of literature on the role of carnitine in animal metabolism, its role in plant metabolism has been less well studied. This chapter reviews work carried out on carnitine in plants and discusses its possible roles in plant metabolism, in particular the shuttling of activated acyl groups between membranebound organelles.

General background

Carnitine (3-hydroxy-4-N-trimethyl ammonium butyrate) is a highly polar compound that is widely distributed in nature. It was discovered over 85 years ago independently by two groups, Kutscher (1905) and Gulewitsch & Krimberg (1905). The latter group found the empirical formula to be C7H15NO3. Crawford & Kenyon (1927) identified its structure as being (CH3)3N+CH2CHOHCH2COO-. Carnitine has two lowenergy conformers; an extended conformer (Fig. L4) and a folded conformer (Fig. IB). Theoretical data show that the preferred conformation for carnitine is the extended form (Murray et al., 1980).

The carbon atom at the 3 position is an asymmetric carbon atom; thus, carnitine exists as D- and L-optical isomers, the L-form being the naturally occurring, biologically active isomer.

After being considered for many years as merely a constituent of vertebrate muscle, a role for carnitine was established when Fraenkel & Blewett (1947) demonstrated that it is an essential growth factor for larvae of the mealworm, Tenebrio molitor. Parallel investigations by Bremer's group (Bremer, 1983) and Fritz's group (Fritz, 1963) established a role for carnitine in the P-oxidation of long-chain fatty acids. In mammals carnitine is not metabolised, except by bacteria in the gut (Bieber, 1988).

In a green plant, cell metabolism is highly compartmentalised. Photosynthesis and photorespiration involve the participation of three different organelles: chloroplasts, mitochondria and peroxisomes. Mitochondria and chloroplasts are surrounded by two membranes; the outer membrane is freely permeable to small molecules like metabolites (Pfaff et al., 1968; Heldt & Sauer, 1971), owing to the presence of pores formed by porins. In chloroplasts these pores allow the passage of substances up to a molecular weight of 10000 (Flügge & Benz, 1984), whereas an exclusion limit of 4000–6000 was found in mitochondria from animal tissues (Zalman et al., 1980). Thus in mitochondria and chloroplasts the inner boundary membranes represent the border between metabolic compartments and are the site of metabolite translocators. Peroxisomes are surrounded by a single membrane. Recent studies have suggested that the boundary membrane of animal peroxisomes also contains porins, allowing the passage of metabolites of molecular mass up to 800 Da (van Veldhoven et al., 1987).

This raises the question, to what extent and by what means peroxisomal metabolism is compartmentalised. This chapter presents a summary of our current knowledge of the processes by which metabolites are transferred between different subcellular compartments and between cells in the course of photosynthetic metabolism.

In any discussion of plant cell metabolism it is essential to recognise the heterogeneity that exists within an organ such as the leaf. This encompasses the variety of cell types, such as the mesophyll, vascular, epidermal, etc., and also the range of cell ages within a leaf. The metabolic role of the organelles may therefore be quite different depending on the cell in which they are localised and also on the developmental stage of that cell. In this chapter we discuss the changes which occur in the size, frequency and metabolic activity of organelles during leaf cell development and differentiation.

Patterns of leaf cell development

Leaf cell development is most readily studied in grasses, such as wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.). This is because all of the cells of the leaf originate from a single meristematic region at the leaf base (intercalary meristem). Thus a developmental gradient is generated whereby the youngest cells are always at the leaf base and there is a measurable range of increasing cell age towards the tip of the leaf. Because of its simplicity, the developing light-grown wheat leaf has been a popular tool with which to study chloroplast and photosynthetic development (see, for example, reviews by Leech (1985) and Baker (1985) in this series). In contrast, dicotyledon leaf development is far more complex as there are several growing regions across the lamina (Maksymowych, 1973). The resulting heterogeneity makes it difficult to separate out areas where cell division, expansion and development are occurring within a dicotyledonous leaf.

The role of mitochondria in cellular energy metabolism in both photosynthetic and non-photosynthetic tissues has been the subject of much study. In non-photosynthetic tissues most of the cell's demand for ATP is met by respiration and oxidative phosphorylation whereas in photosynthetic tissues photophosphorylation can be a major contributor to cellular ATP requirements. The pathways involved are central to metabolism and interact with many other metabolic systems. An understanding of the mechanisms that control respiration is therefore vital for a fuller comprehension of cellular metabolism and efficiency. Before attempting to answer the question of what controls respiratory activity it is important to define the system in question and its limits since there are many different levels at which control can be said to occur. Such a definition allows discrimination between internal controls of the system and the effect on the system imposed by external causes.

The scope of this chapter is restricted to the system of oxidative phosphorylation and respiration in plant mitochondria. The general properties and characteristics of plant mitochondria are well documented and the reader is therefore referred to these articles (Douce, 1985; Moore & Rich, 1985; Douce & Neuburger, 1989; Moore & Siedow, 1991) for a fuller description of their structural and functional properties. The system is defined as comprising the electron transport chain, the intramitochondrial NAD+ and phosphate pools, the adenine nucleotide translocator, the ATP synthase, the protonmotive force and the proton conductance of the inner membrane.

The phenomenon of C3–C4 intermediate photosynthesis has now been recognised for about 25 years and has received a considerable amount of interest in that time, much of it aimed at the link that these plants might represent in the evolution of the C4 photosynthetic system from the older C3 one. A number of recent reviews have addressed evolutionary and adaptive aspects of C3–C4 photosynthesis together with the biochemistry (Peisker, 1986; Edwards & Ku, 1987; Monson, 1989; Monson & Moore, 1989; Araus et al., 1991). We intend, therefore, to confine our discussion in this article to those aspects of the C3–C4 character that have a major influence on the metabolic interactions within and between photosynthetic cells in the leaves of these plants and to compare and contrast this with what is known about photosynthetic metabolism in C3 and C4 plants.

Plants which have C3–C4 intermediate photosynthesis have been identified in seven genera across five families, including representatives from the Monocotyledoneae and the Dicotyledoneae (Edwards & Ku, 1987). Despite their wide distribution in the higher plant kingdom there are a number of well-conserved features across this group. We will describe these and then discuss their implications for inter- and intracellular metabolite transport and the metabolic regulation of photosynthesis and photorespiration.

Summary

Double fertilisation in angiosperms remains an enigma. The two male gametes from the pollen grain or tube fuse with the two female gametes in the embryo sac included in female tissues. Structural data in Plumbago showed that the plastid-rich male gamete preferentially fuses with the egg cell leading to the embryo (Russell, 1985). In maize BMS line, the male gamete bearing the non-disjointed B-chromosome preferentially fuses with the egg cell (Roman, 1948). Is this preferential fusion the result of recognition events at the gamete level? In order to answer this question, one needs to work with male and female gametes isolated from their gametophytes. For this purpose, we chose Zea mays (maize) as a plant model, because it offers large amounts of pollen and female flowers. We succeeded in isolating intact, viable and functional gametes (Dupuis et al., 1987; Wagner et al., 1989b; Roeckel, 1990). What, then, is the best strategy for intergametic recognition studies? We decided to construct a library of monoclonal antibodies directed against cell surface determinants of isolated male gametes. These immunological probes will be used to assess membrane dimorphism of the two male gametes of one pollen grain. We also attempted to develop an in vitro model of intergametic fusion. Experiments of fusion inhibition by monoclonal antibodies in this in vitro model would allow us to sort specific cell surface determinants involved in gametic recognition.

Introduction

The process of pollination and fertilisation in flowering plants involves a series of interactive events between male and female cells. One of the earliest stages in the process of fertilisation is the recognition, and acceptance or rejection, of pollen grains alighting on the stigma of the recipient plant. Self-incompatibility (SI) involves these processes. Prevention of self-fertilisation is accomplished by the inhibition of pollen that has the same incompatibility phenotype as that of the stigma on which it lands. These highly specific recognition events are both developmentally expressed and tissue-specific. Investigation of the molecular basis of the expression and regulation of the S-genes, and the mode of action of their products, therefore, provides a model system for the study of gene expression and cellular recognition in flowering plants.

There is currently considerable interest in the elucidation of the molecular basis of SI and much work has been carried out in an attempt to identify the molecules involved in this interaction, especially those on the female side. S-linked glycoproteins from styles and stigmas, and the genes that encode them, have been identified and cloned. Less progress has been made with the pollen component. We aim to look at what is currently known about SI, with a view to examining what is known about the mechanism of this response.

What is known about the pistil and pollen components?

Identification and characterisation of stigmatic S-linked glycoproteins

There have been a number of studies carried out on proteins which have been isolated from stigmatic/stylar tissues.

A principal feature of plant growth is the maximisation of surface area. This arises from the need of a sedentary organism to obtain the full spectrum of nutrients from the environment. One consequence of this survival strategy is increased vulnerability to pathogens and adverse conditions, since the subterranean and aerial boundaries of the organism with the external world will be immense.

Given this immensity and the lack of any specialised surveillance cells equivalent to the mammalian immune system, cells throughout the organism have evolved an ability to recognise foreign from self. The results of these molecular recognition events are reflected at the local site of stimulus perception but, importantly, are also transmitted to distant regions of the plant. There is now good evidence that defence gene expression and changes in the levels of defence-related products such as phytoalexins, callose and lignin are modulated by these local and systemic signalling events.

This chapter reviews research carried out on these topics at the University of Leeds, with particular reference to (1) plant defence responses to parasitic nematodes and (2) the molecular effectors of the wound-response. General literature to 1990, on defence-related proteins in higher plants has been reviewed by Bowles (1990a).

Plant–nematode interactions

One of the plant–nematode systems under study in my laboratory at Leeds University involves the response of potato (Solanum tuberosum) plants to potato cyst nematodes (Globodera spp.). The potato cultivar Maris Piper carries a single dominant gene for resistance (H1) that is effective against certain pathotypes of Globodera rostochiensis (for example, Ro1), but is ineffective against others (for example, Ro2) and against the closely related Globodera pallida (Sidhu & Webster, 1981).

Introduction

The host–parasite interface formed during infection of plants by biotrophic powdery mildew fungi is a specialised structure involved in the transfer of host nutrients to the fungus (Manners & Gay, 1983; Manners, 1989) and the efficiency with which such pathogens do this makes them highly damaging to crops (Singh et al., 1982). The haustorium is the structure by which the fungus absorbs nutrients from its host. It forms within the epidermal cells, enclosed by an invagination of the host plasma membrane termed the extrahaustorial membrane (ehm); this is separated from the haustorium by the polysaccharide-rich extrahaustorial matrix. All these components collectively make up the haustorial complex (HC; see Fig. 1.).

Powdery mildew fungi are obligate pathogens and exhibit a high degree of host species and cultivar specificity. In certain incompatible combinations of host and pathogen genotype, haustoria are produced but appear to be functionally inactive and so fail to produce elongating secondary hyphae or secondary haustoria (e.g. Erysiphe pisi (Singh & Singh, 1983; Manners & Gay, 1983; Manners, 1989); Erysiphe betae (Dickey & Levy, 1979)). Work on the E. pisi/Pisum sativum system using various cytochemical reagents such as fluorescent lectins, as well as protein and Ca2+-specific fluorochromes has shown that there may be molecular differences between the ehm of haustoria from resistant compared with susceptible interactions (Chard & Gay, 1984). It is therefore a possibility that the molecular recognition events, mediated by the primary products of host genes for resistance and pathogen genes for avirulence (Callow, 1984, 1987) and which are regarded as the first step in resistance triggering through intracellular signalling, are located at this interface.

Introduction

Plant roots generate electrical currents and voltages in the rhizosphere that may influence the behaviour of the many pathogenic, symbiotic or commensal microorganisms that live in association with them. These electrical currents represent circulations of protons and other ions. Consequently, they also lead to the creation of substantial ionic and pH gradients whose affect on the infection and colonisation of the root is only now being explored. Here, we summarise briefly what is known about the electrical currents of plant roots and discuss the ways in which they may influence the root microflora. In particular, we focus on the swimming zoospores of Phytophthora species, which are exquisitely sensitive to electrical fields and may target their host roots using a combination of chemotaxis and electrotaxis.

Growth and electricity

It has been known for many years that plants generate electrical currents (Müller-Hettlingen, 1883; Lund & Kenyon, 1927; Lund, 1947). These were first measured using microelectrodes inserted into cells at different regions of a root or tissue. Voltage differences were found between different sites and electrical current was presumed to flow through the cells between the electrodes and through the extracellular medium to complete the circuit. Endogenous currents in the extracellular loop of the circuit can now be measured directly, without invading cells with intracellular microelectrodes. In the 1970s ultrasensitive, voltage-sensing vibrating electrodes were devised that are capable of detecting the minute electrical fields generated by individual cells or tissues (Jaffe & Nuccitelli, 1974).

Summary

Many fungal pathogens use contact sensing of the host surface to achieve successful infection. Contact-mediated responses can be induced and experimentally examined on artificial substrata which mimic physical characteristics of the host surface. The necessary physical and spatial characteristics of surfaces which induce contact-mediated responses are described and classified as either topographical or non-topographical. Topographical signals may be in the form of steps, ridges or furrows and these sometimes require very precise dimensions and/or spacings to induce a fungal response. Non-topographical signals lack this specificity because the fungus recognises only that it is in contact with a surface possessing suitable physical features. Examples of contact-mediated responses include the asymmetric organisation of a cell relative to its substratum, adhesion of a cell to the contact surface, changes in spore surface morphology and accompanying release of enzymes, directional growth of hyphae, and induction of appressorium differentiation. Evidence of these processes being contact-mediated is assessed and possible mechanisms by which fungal contact sensing may operate are discussed. Finally, strategies for controlling plant diseases through an understanding of contact sensing are defined.

Introduction

A large number of eukaryotic cells are sensitive to contact (touch) stimuli which affect their behaviour, growth and morphogenesis in a variety of ways. Numerous examples of contact sensing by animal and plant cells have been reported (see e.g. Braam & Davis, 1990; Curtis & Clark, 1990; and references cited therein). Contact sensing also plays an important role in the infection of plants by many fungal pathogens.

This volume is a collection of papers presented at the ‘Perspectives in Cell Recognition’ Seminar Series symposium during the annual SEB conference held at Birmingham University, UK, in April 1991. The basic purpose of the sessions was to bring together biologists working on diverse aspects of recognition in plants to discuss recent progress in our understanding of ‘self’ and ‘non-self’ interactions between cells. The two major areas of biological interactions covered were those involving dissimilar cells of the same organism associating as gametes in sexual reproduction, and those involving cells of different or ‘foreign’ organisms, associating in either pathogenesis or mutualistic symbioses. It is in these areas that greatest progress has been made in understanding the cellular, molecular and genetic mechanisms involved and in some cases, notably in the Rhizobium–legume symbiosis, the actual genes that control specificity and which are involved in creating a harmonious mutualism have been cloned and their products characterised. Also included are contributions on the exciting progress that is being made in characterising the surface glycoproteins of higher plants that are involved in associations between somatic cells that are crucial to coordinated tissue development, and aspects of cell–cell communication involved in the systemic responses of plants to wounding or pathogenic stimuli.

It was, of course, tempting to include contributions on many other aspects of recognition, such as work on plant hormone receptors and environmental cues, and the intracellular signalling responses associated with recognition.

Introduction

In the unicellular green alga Chlamydomonas the two flagella are used not only for locomotion but also for sexual cell–cell interactions. In a compatible combination of mating-type plus and minus (mt+ and mt−) partners, the cells adhere to each other by their flagella. This results not only in a close proximity of the cell bodies that are going to fuse with each other, but also in the generation of a signal, telling the cells to prepare for fusion. This implies at least the total or partial hydrolysis of the cell wall, and the activation of a specialised zone at the anterior part of the cell surface. Thus, Chlamydomonas is an example in which signalling is generated by physical cell-to-cell contact. This allows us to investigate the nature and behaviour of the surface receptors involved in this process and the mechanism of signal transduction over the surface membrane and inside the cells. Despite the clarity in which these processes in this simple eukaryotic system are exhibited, and the considerable progress that has been made in a number of laboratories, several enigmas remain, which I address in this chapter.

Strategies of gametic approach

Most of the research concerning sexual interactions has been carried out with the heterothallic Chlamydomonas eugametos and Chlamydomonas reinhardtii. In these species, cells within one clone all have the same mating type. So when mt+ and mt− clonal populations are mixed, the cells exhibit sexual conjugation when they are mating-competent.

Abstract

Recognition between leguminous plants and the specific rhizobial strains that nodulate them is mediated via a regulon of nodulation (nod) genes present in the bacteria. These nod genes are induced by flavonoids secreted from legume roots. Many of the nod gene products are involved in the synthesis of host-specific signals that are recognised by appropriate legume hosts. Recently (Lerouge et al., 1990), the signal molecule made by one strain of Rhizobium meliloti was identified as an acylated and sulphated, tetraglucosamine glycolipid and there is strong evidence that Rhizobium leguminosarum makes related but structurally distinct signals.

On the basis of these observations it is now possible to make sense of several similarities that have been recognised between nod gene products and enzymes of known function. Thus, for example, it appears that the nodM gene product is involved in the formation of glucosamine precursors of the signal molecule, whilst other gene products are likely to be involved in specific substitutions that confer host specificity to the signal molecule.

In addition to those nod gene products that are involved in the synthesis of the glycolipid, it is evident that there are other genes which may carry out a different role. Of particular interest is the nodO gene which encodes a secreted Ca2+-binding protein that has the potential to interact directly with plant cells. In the absence of the nodFEL genes, nodO is necessary for nodulation, indicating that the NodO protein can compensate for the loss of nodFEL function during infection.