Introduction

In this chapter, I discuss the current state of knowledge about the biochemistry and molecular biology of phosphate starvation inducible (psi) enzymes and proteins in higher plants. Special attention will be paid to the excreted phosphate starvation inducible (epsi, pronounced ee Ψ) acid phosphatase (APase) of higher plants. A significant amount of data now exists to support the theory that the epsi-APase, an epsi-RNase, several intracellular RNases and other proteins of unknown function form a family of co-induced proteins that act, at least in part, as a phosphate starvation rescue mechanism for higher plants. In addition, we have conducted experiments to show that some of these proteins can apparently affect phosphate use-efficiency metabolism under non-starvation conditions. While crucial experiments remain to be done, our data further suggest that the epsi-APase genes as well as genes for other psi proteins can be regulated at the mRNA level (possibly by transcriptional activation). We have proposed, as a working model, that these psi genes are part of the higher plant pho stimulon and may be co-regulated by the same trans-acting element(s) to form a pho regulon (Goldstein et al., 1989a).

Phosphorus is an essential nutrient for all cells. For organisms that absorb their mineral nutrients directly from the external medium, ionic inorganic phosphate (Pi, usually H2PO4− or HPO42−) is the preferentially absorbed form of phosphorus. A macronutrient based on its contribution to biomass, Pi is one of the least available mineral nutrients in many environments.

Introduction

Nitrogen is the most common limiting factor in the growth and productivity of plants. Legume plants, however, have overcome this limitation by developing the ability to harbour a group of soil bacteria (Azorhizobium, Bradyrhizobium, Rhizobium and Sinorhizobium spp.) in a symbiotic association. In this mutually beneficial interaction, bacteria invade the root cells of host plants where they become intracellular ‘organelles’ called bacteroids and are able to fix atmospheric dinitrogen into ammonium for assimilation by the plant. To facilitate this process, the host plant develops an entirely new organ, a root nodule, which houses the nitrogen-fixing bacteria and provides the carbon sources, other nutrients and appropriate environment to support the reduction of dinitrogen (see Verma & Long, 1983; Long, 1989; Verma & Stanley, 1989).

The Rhizobium–legume symbiosis, an interaction between a prokaryote (Rhizobium) and a eukaryote (legume), requires a series of sequential induction and function of both bacterium-encoded (bacteroidins) and host-encoded (nodulins) nodule-specific proteins. It has been shown that many plant (Peters et al., 1986; Firmin et al., 1986; Djordjevic et al., 1987; Sadowsky et al., 1988; see also Peters & Verma, 1990) and bacterial (Lerouge et al., 1990; Kondorosi, 1991) signals are involved in the induction of specific genes leading to the development of the root nodule. Many bacterial mutations blocking root hair deformation, induction of cortical cell division, infection thread formation and subsequent events leading to the endocytotic release of bacteria inside the host cell are known (see Rolfe & Shine, 1984).

Introduction

The growth of plants in nature depends on their ability to respond to their environment. For the metabolism of metals, plants require a balance between the uptake of sufficient essential metal ions to maintain growth and development and the ability to protect sensitive cellular activity from excessive concentrations of essential and non-essential metals. Although phytotoxic amounts of metal occur more frequently from industrial and agricultural pollution than in soils under natural conditions, nevertheless, survival mechanisms are required to detect not only external/internal concentrations of metals, but also essential from non-essential metal ions. Plants thus have the ability to ‘sense’ metal ions since it is central to normal metal metabolism, protection from metal toxicity, and adaptation to metal tolerance. Such recognition can be envisaged to occur by a number of physiological processes, but at the molecular level it is likely to be the binding of metal ions to a protein, which directly or indirectly changes the pattern of cellular activity, usually by changing gene expression. Evidence is now emerging that this molecular recognition is ‘programmed’: evolution has fashioned proteins either to have rigid binding sites which accept some ions while rejecting others, or to have flexible binding sites in which the stereochemistry of the ion determines the final shape of the protein. In either case, evolution has given the organism the capability to distinguish metals and partition them in different ways.

Introduction

Acquired resistance in plants has long been recognised to play an important role in the preservation of plants against disease (Chester, 1933). While much of this early work focused on viral cross-protection, the plants' ability to induce a defence against disease became a topic of research by the early 1960s (Ross, 1961a,b). In these studies tobacco mosaic virus (TMV) was inoculated onto the leaf of a tobacco variety that produced necrotic lesions. Seven days after infection, at a time when lesions had formed on the leaf, both the inoculated leaf and uninfected leaves on the same plant had become resistant to further infection by TMV. The resistance was directed not only against TMV but also other unrelated viruses, as well as certain fungal and bacterial pathogens. Further, the resistance could be induced by other pathogens including viruses, bacteria and fungi, the only common requirement being the development of some necrosis from the infection. Ross referred to the resistance in infected leaves as localised acquired resistance (LAR: Ross, 1961a) and the resistance that developed in the uninfected leaves as systemic acquired resistance (SAR: Ross, 1961b). Although resistance to virus was the main topic of these studies, later emphasis has been on the non-specificity and broad spectrum of SAR against various fungal and bacterial diseases (Hecht & Bateman, 1964; Kuc, 1982; Dean & Kuc, 1985).

A number of exogenously applied chemicals, including polyacrylic acid, acetylsalicylic acid, salicylic acid and isonicotinic acid (NA) derivatives, have also been shown to induce resistance.

The anaerobic response

When plants experience anoxic conditions there is a shift in carbohydrate metabolism from an oxidative to a fermentative pathway (Fig. 1). In the absence of oxygen, ATP is generated not by the Krebs cycle but by alcoholic fermentation, i.e. glycolysis and ethanol synthesis.

As well as the change in carbohydrate metabolism there is a change in the pattern of polypeptide synthesis under anoxia (Sachs et al., 1980; Bailey-Serres et al., 1988). Synthesis of polypeptides normally present under aerobic conditions stops and synthesis of a number of specific polypeptides – the anaerobic polypeptides (ANPs) – commences. In maize there are about 20 ANPs which have been identified chiefly as enzymes associated with the flow of carbon into and through glycolysis and through alcoholic fermentation; in particular UDP-sucrose synthetase, pyruvate decarboxylase and alcohol dehydrogenase (ADH) are induced approximately 10-fold (Lazlo & St Lawrence, 1983; Springer et al., 1986). Glucose phosphate isomerase (Kelley & Freeling, 1984a), one of the isozymes of glyceraldehyde 3-phosphate dehydrogenase, and cytoplasmic aldolase (Kelley & Freeling, 1984b) have also been shown to be induced to a lesser degree. The levels of two enzymes which are thought to be responsible for regulating the glycolytic pathway, phosphofructokinase and pyruvate kinase, do not change significantly during anaerobiosis (Bailey-Serres et al., 1988).

It is assumed that the enzymes of glycolysis are induced by anaerobiosis to allow a greater flux of carbohydrate through the pathway because only 2 molecules of ATP are produced per molecule of glucose under anaerobic conditions whereas 36 molecules of ATP are produced under oxidative conditions.

Introduction

Plants growing in temperate climates are often exposed to cold stress which can kill the plants. The cold or low temperature stress can be subdivided into chilling (temperatures above 0°C) and freezing (subzero temperatures) stress. The absolute temperatures which lead to damage in plants vary markedly between different plant species. Cold resistance measured as the frost killing point is not a constant for a genetically pure variety but is strongly influenced by environmental factors and the developmental stage of the plants.

Two basic mechanisms to survive a low temperature stress have evolved in plants: either to avoid the fatal temperature or to adapt to the low temperature by developing tolerance towards the stress. Although the plant is essentially unable to avoid the freezing temperature of its environment, it is suggested by Levitt that some protection can be obtained by supercooling or accumulation or antifreeze as a mechanism for avoidance (Levitt, 1980). On the other hand, many temperate plant species have the capability of cold acclimation: exposure to a period of low, non-freezing temperatures can lead to a significant increase of cold tolerance (Levitt, 1980). This process is termed cold acclimation or cold hardening. The interrelationship of these different processes is shown in Fig. 1.

The subject of this chapter will be to summarise the biochemical and molecular changes which take place during the process of cold acclimation and the acquisition of freezing stress tolerance.

Biological principles of fruit and seed production

The life cycles of flowering plants can be generalized, with a few exceptions in which sexual reproductive capacity has apparently been lost to clonal vegetative reproduction or distorted by such devices as apomixis (Fig. 1). Within this simple framework, however, the variation is enormous: the duration of the life cycle may range from days to centuries, and the proportion of the biomass invested in reproduction varies with species, genotype, age of the plant and environmental conditions (Baker, 1972). For a given reproductive investment, species may trade off large numbers of tiny seeds from a single flower, as in many orchids, as against a few bulky seeds with large reserves from a large number of flowers, as in the pome fruits. There is abundant evidence that the evolution of these details of plant reproductive biology has been influenced by a wide variety of physical factors such as availability of nutrients, light and water (Mooney, 1972) and biological factors such as the nature, availability and energetics of pollinators (Heinrich, 1975), the agents of fruit and seed dispersal (Gautier-Hion et al., 1985) and the activity of pathogens and predators (Janzen, 1977). One may trace in the literature of plant reproductive biology a process of gradual description and elucidation of factors which may influence reproductive behaviour (Lloyd, 1980; Lloyd, Webb & Primack, 1980; Sutherland, 1986; Primack, 1987; Stephenson, Devlin & Horton 1988) and a steadily increasing sophistication in the ‘telling of adaptive stories’ which purport to ‘explain’ observed behaviour (Gould & Lewontin, 1979).

Flowering and fruiting are the key processes in the biology of higher plants that ensure the transfer of genetic material from one generation to the next. Furthermore, almost the whole of the world's agricultural and horticultural industries depend upon the production of flowers, fruits and seeds, and so the reproductive biology of cultivated plants is of fundamental importance to humankind. However, it is surprising that compared with studies on the growth and development of vegetative structures, reproductive biology seems to have received somewhat less attention from environmental physiologists.

Previous meetings of the Environmental Physiology Group of the Society for Experimental Biology have considered various aspects of vegetative growth in some detail and these have resulted in SEB Seminar Series publications on The Control of Leaf Growth (Cardiff Meeting, 1984), Root Development and Function (Bangor Meeting, 1985) and on Plant Canopies (Nottingham Meeting, 1986). It thus seemed timely to devote a meeting to various aspects of reproductive growth and development and so this became the subject of the 1990 meeting of the Environmental Physiology Group at the Society's annual conference at University of Warwick. It was agreed from the outset that this topic should be approached on a broad front, from the onset of flowering to the development and growth of fruits and seeds, and finally to ecological and evolutionary aspects of fruiting. Thus future meetings of the Group can focus more narrowly on topics such as seed growth and development.

Introduction

It is a widely accepted principle of plant geography that the distribution of vegetation and of plant species is primarily controlled by climate, but that climate changes with time (Cain, 1944). The basis of this principle is the broad correlation of vegetation with latitude, essentially a correlation with temperature, and the modification of this relationship by the availability of water.

These correlations are well illustrated by the boundaries of the climax woodlands of northern Europe, which run latitudinally across northern Russia and Finland but then have a southwestern trend across Scandinavia and Britain (Sjörs, 1965). These boundaries are correlated with various measures of summer warmth. The northern limits of many of the constituent species are similarly correlated with summer warmth but also with the vegetational zones themselves.

The possibility that temperature controls the boundaries through its effect on reproduction rather than vegetative growth is suggested from the common observation that heavy crops of fertile fruit of some of the dominant species of tree follow, or coincide with, years of exceptional warmth (Matthews, 1955). There is, however, very little information about the production of fertile fruit at the northern limits of species, nor are there many studies of the population dynamics of species at their limits, so that the relation between reproductive capacity and regeneration is unknown.

Studies of species in northwestern England

Recent studies on the distribution of vegetation in Britain for the National Vegetation Classification (Rodwell, 1991) provide many examples of plant communities that are restricted either to the warmer and drier southeast or to the cooler and wetter northwest of Britain.

Introduction

Fruit growth is part of the integrated growth of a plant. Therefore, fruit yield is determined by the interaction between growing conditions and morphological characters, as well as the physiological activities, of the whole plant.

It has long been recognized that the improvement of fruit yield is dependent on our understanding of the factors controlling both the production by the leaf (i.e. assimilate production) and the sink strength of the fruit (i.e. assimilate partitioning) (see Watson, 1968). Most likely, the key to understanding the regulation of fruit growth is to identify the responses to the environment by the morphological factors and metabolic processes inside the fruit interacting with those in the rest of the plant. In terms of assimilate partitioning, fruits are irreversible storage sinks, as the imported assimilate is either used for growth or stored as reserves and no net export occurs during the life of the organ (Ho, 1988). Therefore, it is essential to know how the supply, or the competition for the supply, of assimilate by the individual fruit is regulated. In this review, I examine fruit growth in terms of source-sink interaction, sink competition and sink strength determination. Evidence will be presented that, apart from the supply of assimilate, both cell number (i.e. sink size) and some of the physiological activities (i.e. sink activity) within a fruit may determine its sink strength in attracting assimilate to sustain fruit growth.

Introduction

In higher plants, fertilization of the ovule leads to the formation of the embryo. Zygotic embryogenesis, the way in which the fertilized ovule develops, has been studied extensively; changes that occur have been described for many plant species at the level of morphology, metabolism, protein composition, and gene expression. Interestingly, plant embryos can also develop in the absence of a fertilized ovule, from somatic cells in callus culture (Sung et al., 1984), from differentiated cells such as leaf mesophyll (Conger et al., 1983) and, perhaps most surprisingly, from immature haploid male gametes termed microspores (Nitsch, 1969; Dunwell, 1985). These alternative routes to the formation of an embryo illustrate both the means by which plants use environmental stimuli as developmental signals and the plasticity of plant development that is maintained throughout growth.

This article is not a comprehensive review of embryogenesis but rather a brief introduction to some interesting key issues in the area, and an outline of the ways in which we have been approaching the subject at Leeds. Two principal issues about plant embryogenesis will be discussed. The first concerns embryo formation, and the second concerns the way in which an embryo, fully capable of germinating within days of organ primordia differentiation, is nevertheless prevented from doing so until seed development has been completed.

Introduction

Seeds contribute on a world basis some 55% of our daily per capita protein and energy supply. Of this, 90% is accounted for by cereals (Duffus & Slaughter, 1980). The contribution is even greater if allowance is made for the inclusion of cereals and other seed crops in the diet of farm animals. It is therefore not surprising that there has long been an interest in the mechanisms regulating seed growth and maturation, since such studies could lead to the identification of key factors involved in the control of yield and quality in the harvested material.

The aim of this chapter is, then, to review present understanding of the control mechanisms involved in seed growth and development. The major emphasis is on the cereals, including barley, wheat, rice, oats and maize. The morphological, physiological and biochemical changes accompanying grain growth and development are described, and the influence of genotype and environmental conditions on these changes is discussed.

Developmental morphology

The cereal seed is surrounded by a seed coat or testa, which is fused to the pericarp, forming a single-seeded fruit known as a caryopsis. The harvested material is termed a grain or kernel; in wheat, rye, triticale, maize, grain sorghum and the naked-grain millets, this consists solely of a caryopsis. In oats, barley, rice and the husked types of sorghum and millet, the grain consists of a caryopsis, together with the lemma and palea, which adhere to the outer surface.

Introduction

In many species, there is a long period from flower induction to fruit ripening and abscission, during which environmental factors can have a major impact on the success of fruiting. This is particularly so with perennial crops such as pome, stone and citrus fruits, where even the period just from anthesis to ripening can last up to 60 weeks (Valencia orange). It is not surprising, therefore, that there are several opportunities for adverse environmental conditions to lead to reduced fruit numbers at harvest. For example, flower abortion before anthesis can result from low light levels (e.g. tomato) (Kinet et al., 1978), frost damage (e.g. pome fruits) (Modlibowska, 1964), water deficit (e.g. Vicia faba L.) (El-Nadi, 1969) and water excess (e.g. Vicia faba L.) (Smith, 1982), while shading can precipitate the drop of enlarging fruits (e.g. peach and apple) (Byers et al., 1985) and wind can exacerbate the pre-harvest drop of pome fruits. Flower and fruit abortion has been reviewed by Stephenson (1981) and will not be the major topic of this chapter.

Although environmental factors can affect fruiting pre-anthesis and after the initial set has taken place, by far the most important period determining the success of fruiting comprises the few days during and after flower opening, when the transition from flower to fruitlet takes place. This, of course, is not surprising since it is also the time when the reproductive competitiveness of the maternal genome is partly determined through the success of fertilization and subsequent seed dispersal.

Environmental control of flowering

The simple observation that many plants flower at a particular season or time of year implies that flowering is influenced and perhaps controlled by changes in the environment. All plants require conditions in which they can grow and develop in order to reproduce and so the ultimate environmental controls are those that determine plant distribution, and the main one may very often be temperature (Grace, 1987). But in all habitats, variations in the environment provide potential cues for the plant to make use of, so that the transition to reproductive growth coincides with the conditions most likely to lead to successful completion of flowering, fruiting and seed dispersal. Even in plants that do not respond to specific environmental changes, the onset and rate of progress of reproductive development will be determined by the general environment of the plant.

Are there any habitats in which the environment is constant and optimum so that growth and reproductive development are governed entirely by factors internal to the plant? The nearest approach to this is probably the tropical forest of SE Asia, which is virtually non-seasonal. However, even here flowering can be controlled by environmental changes, but because these are infrequent, mass flowering is also infrequent. When it does occur, up to 88% of the species may flower simultaneously (Appanah, 1985). The close correlation between an environmental change and subsequent flowering has been shown for the rain forest in Singapore, one of the least seasonal places in the world (Corlett, 1990).