Introduction to Ecological Biochemistry

By J. B. Harborne

Ecological biochemistry concerns the biochemistry of interactions between animals, plants and the environment, and includes such diverse subjects as plant adaptations to soil pollutants and the effects of plant toxins on herbivores. The intriguing dependence of the Monarch butterfly on its host plants is chosen as an example of plant-animal coevolution in action.

The ability to isolate trace amounts of a substance from plant tissues has led to a wealth of new research, and the fourth edition of this well-known text has consequently been extensively revised. New sections have been provided on the cost of chemical defence and on the release of predator-attracting volatiles from plants. New information has been included on cyanogenesis, the protective role of tannins in plants and the phenomenon of induced defence in plant leaves following herbivory.

Advanced level students and research workers aloke will find much of value in this comprehensive text, written by an acknowledged expert on this fascinating subject.

• The book covers the biochemistry of interactions between animals, plants and the environment, and includes such diverse subjects as plant adaptations to soil pollutants and the effects of plant toxins on herbivores
• The intriguing dependence of the Monarch butterfly on its host plants is chosen as an example of plant-animal coevolution in action
• New sections have been added on the cost of chemical defence and on the release of predators attracting volatiles from plants
• New information has been included on cyanogenesis, the protective role of tannins in plants and the phenomenon of induced defence in plant leaves following herbivory

• Language: English
• Publisher: Academic Press
• Release date: Jun 28, 2014
• ISBN: 9780080918594

Book preview

1977

Preface

The last two decades have witnessed the growth of a new inter-disciplinary subject, variously termed ecological biochemistry, chemical ecology or phytochemical ecology, which is concerned with the biochemistry of plant and animal interactions. Its development has been due in no small measure to the increasingly successful identifications of organic molecules in microquantities, following the application of modern chemical techniques to biological systems. It has also been due to the awareness of ecologists that chemical substances and particularly secondary metabolites such as alkaloids, tannins and terpenoids have a significant role in the complex interactions occurring between animal and animal, animal and plant or plant and plant in the natural environment. A further stimulation has been the possible applications of such new information in the control of insect pests and of microbial diseases in crop plants and in the conservation of natural communities. The present text is intended as an introduction to these new developments in biochemistry that have so enormously expanded our knowledge of plant and animal ecology.

This book is based on a course taught by the author over a number of years, both at Reading and at other universities. It has been planned so that it is suitable for second or third year university teaching in Departments of Botany, Biochemistry and Biological Sciences. Two general points may be emphasized about the use of this text for university teaching. First, each chapter is intended to be self-contained, so that there should be no problem if the order is rearranged to meet the requirements of a particular course. Second, in the bibliography of each chapter, the major books and review articles have been separated from the other references with the intention that these might form a reading list for students. It is also hoped that the book will be of more general value as a simple introduction to a new subject area.

In preparing this fourth edition, every effort has been made to bring it up to date with the latest developments in the subject. New sections have been provided on the cost of chemical defence and on the release of predator-attracting volatiles. The section on increased synthesis of toxins has been rewritten. A summary has been added to Chapter 3 and new references have been included in many places.

The author is grateful to Dr Miriam Rothschild, FRS for her introductory foreword. By her own pioneering experiments with aposematic insects and equally her encouragements of other scientists, Dr Rothschild has contributed more than anyone else to this new subject and this book owes much to her example, The author is also grateful to many friends and colleagues who have provided him with reprints of their work in this field. He also thanks Miss Valerie Norris, his secretary, for her assistance in revising the text. Finally, he has pleasure in acknowledging his debt to the editorial staff of the publishers for their enthusiasm and expertise in handling this venture.

This edition, like the third, is dedicated to the memory of Professor Tony Swain, 1922-1987, a close friend and colleague, and one of the major pioneers in the development of the subject of ecological biochemistry.

March 1993

Jeffrey B. Harborne,     University of Reading

1

The Plant and Its Biochemical Adaptation to the Environment

Publisher Summary

This chapter presents an account of the explosive development in ecological biochemistry that has occurred in the last two decades. Ecology is largely observational, is concerned with interactions between living organisms in their natural habitats, and is carried out in the field. By contrast, biochemistry is experimental, is concerned with interactions at the molecular level, and is carried out at the laboratory bench. Nevertheless, these two distinctive disciplines have cross-fertilized in recent years with astonishing success and a whole new area of scientific endeavor has opened up as a result. To the ecologist, knowledge of biochemistry has illuminated to a remarkable degree the complex interactions and co-evolutionary adaptations that occur between plant and plant, plant and animal, and animal and animal. Similarly, to the biochemist, studies in ecology have provided for the first time a rational and satisfying explanation for at least a part of the enormous proliferation of secondary metabolism that is observed in plants. The chapter discusses the types of environmental factors that plants are subject to. It also discusses the biochemical bases of adaptation to climate, saline habitats, soil, and high concentrations of toxic metals in soils.

I Introduction

II The biochemical bases of adaptation to climate

A General

B Photosynthesis in tropical plants

C Adaptation to freezing

D Adaptation to high temperatures

E Adaptation to flooding

F Adaptation to drought

III Biochemical adaptation to the soil

A Selenium toxicity

B Heavy metal toxicity

C Adaptation to salinity

IV Detoxification mechanisms

A General

B Detoxification of phenols

C Detoxification of systemic fungicides

D Detoxification of herbicides

V Conclusion

Bibliography

I

Introduction

The marriage between such diverse disciplines as ecology and biochemistry may seem at first a curious alliance. Ecology is largely observational, is concerned with interactions between living organisms in their natural habitats and is carried out in the field. By contrast, biochemistry is experimental, is concerned with interactions at the molecular level and is carried out at the laboratory bench. Nevertheless, these two distinctive disciplines have cross-fertilized in recent years with astonishing success and a whole new area of scientific endeavour has opened up as a result. Ecological biochemistry is only one of a variety of phrases that have been employed to describe these exciting developments.

To the ecologist, knowledge of biochemistry has illuminated to a remarkable degree the complex interactions and co-evolutionary adaptations that occur between plant and plant, plant and animal and animal and animal. It has led to the realization, for example, that plants are functionally interdependent with respect to their animal herbivores and form what are termed ‘plant defense guilds’ (Atsatt and O’Dowd, 1976). Similarly, to the biochemist, studies in ecology have provided for the first time a rational and satisfying explanation for at least a part of the enormous proliferation of secondary metabolism that is observed in plants. Much of the purpose of the synthesis of complex molecules of terpenoids, alkaloids and phenolics lies in their use as defence agents in the plant’s fight for survival against animal depredation.

The aim, therefore, of the present text is to provide an account for the student reader of the explosive development in ecological biochemistry that has occurred in the last two decades. The various chapters deal in turn with the plant and its interactions with animals and with other plants, while animal-animal interactions are considered in some detail in Chapter 8. It should be emphasized at this point that the biochemistry of many interactions has been deliberately simplified here in order to present a coherent story. It must be recognized that a given interaction between a plant host species and its animal predator species can be very subtle and complex and certain aspects of such an interaction may require many years of study before all is revealed. Furthermore, a third organism may have a controlling influence on a plant–animal interaction, such as a parasitoid of the animal or a microbial infection of the plant.

The term plant is generally used throughout this book to refer to higher plants and mainly to angiosperms, gymnosperms and ferns. Fungi, bacteria and viruses will usually be referred to as micro-organisms; other groups of plants will rarely be mentioned—i.e. algae, mosses and liverworts—largely because their ecological biochemistry has not yet been studied in much detail.

The selection of animals mentioned in this text is restricted to those taxa that have been studied experimentally and is certainly very unrepresentative of the animal kingdom as a whole. This is because plant-animal interactions in terms of feeding and defence have largely centred on the insect kingdom and only more recently have biochemical aspects of mammalian ecology been explored to any extent.

The emphasis here on the plant is due, at least in part, to the fact that plants are richer than animals in their biochemical diversity. Although secondary metabolism occurs in animals (Luckner, 1990), nevertheless, over four-fifths of all presently known natural products are of plant origin (Robinson, 1980; Swain, 1974). Some idea of the range of secondary compounds found in plants can be obtained from Table 1.1, which lists some of the major classes, together with an indication of numbers of known compounds, distribution patterns and biological activities. Many of these substances will be mentioned in more detail in subsequent chapters. The richness in secondary chemistry in plants is at least partly explicable by the simple fact that plants are rooted in the soil and cannot move; they cannot respond to the environment in ways open to animals.

Table 1.1

Major classes of secondary plant compounds involved in plant–animal interactions

In animals, secondary metabolism is associated particularly with defence and with signalling and can be very diverse in some instances (see Chapter 8). Animal metabolites do not differ generally from those produced in plants and there are many biosynthetic pathways which are common. The cyanogenic toxins linamarin and lotaustralin, for example, are accumulated by both plants and insects and they are formed in both cases from valine and isoleucine, respectively.

In this first chapter, attention is focused on biochemical adaptation. In its widest sense, this topic continues in later chapters, but here, it is taken in the narrower sense as adaptation to the physical environment. Attention is deliberately restricted to the plant kingdom, where information on biochemical adaptation is only of recent origin. Parallels between plant and animal processes will be mentioned, whenever these are relevant. Much is known about biochemical adaptation in animals and the subject is well documented in textbooks on comparative biochemistry (e.g. Baldwin, 1937; Florkin and Mason, 1960–1964). A useful reference to the subject of biochemical adaptation of animals to environmental change is that of Smellie and Pennock (1976).

Adaptation represents the ability of a living organism to fit into a changing environment, at the same time improving its chances of survival and ultimately of reproducing itself. The extensive diversity of life forms on this planet (i.e. several million species) and their presence in every type of habitat are witness to the view that living organisms indeed are morphologically and anatomically adapted to their environments. Such ideas are fundamental to the Darwinian view of nature and have been supported by much experimentation during the last century. Ideas of physiological and biochemical adaptation came later, during the 1920s and 1930s, with the experimental development of these two subjects. It is only, however, very recently that biochemical aspects have been developed sufficiently with plants to warrant their separate consideration, as in this present chapter.

Adaptation is generally considered as occurring on an extensive time-scale, involving many generations, but it can also take place during the lifetime of an individual, when it is sometimes termed acclimatization. The term adaptation is used here largely in the evolutionary sense. Biochemical adaptation is particularly closely connected to physiological adaptation and indeed it is sometimes difficult to distinguish the two. Physiological adaptation in plants will only be considered here, where appropriate, in relatively brief terms. For a comprehensive account, see Levitt (1980). Two excellent books on the subject for the student reader are Crawford (1989) and Fitter and Hay (1987).

Biochemical adaptation can operate at different levels in metabolism. It may affect the enzymes and produce amino acid substitutions in the primary sequence of protein or else alter the balance of isozymes. It may affect intermediary metabolism; an example in the case of the carbon pathway in photosynthesis is mentioned later. Finally, it may affect secondary metabolism; this is especially true of the plant’s adaptation to animal feeding.

The environmental factors that plants are subject to can be broadly divided into five types:

(1) Climatic factors. These include temperature, light intensity, daylength, moisture and seasonal effects.

(2) Edaphic factors. All plants, except aquatics, epiphytes and parasites, obtain their mineral nutrition through the soil. The soil is also the source of symbiotic microbes, e.g. those required by legumes and other nitrogen-fixing plant species. Through contact with the soil, plants may have to cope with toxic heavy metals or with excess salinity. Equally, they may be subject to biochemical stress due to mineral deficiency in the soil.

(3) Unnatural pollutants. These are distributed through the upper atmosphere (ozone, industrial gases, gasoline fumes) or through the environment (organic pesticides) and may be potentially toxic to many plants.

(4) Animals. Although there is an element of symbiosis in animal feeding, herbivores are primarily hostile to plants, since they depend on them for their very existence. Many different defence adaptations are known in plants. An element of symbiosis is also present in the case of those animals which visit plants for the purpose of pollination (see Chapter 2).

(5) Competition from other plants. This can be either competition between different higher plants or between different forms of plant life, e.g. higher plants and micro-organisms.

In this chapter, we are concerned only with the first three factors: the climate, the soil, and unnatural pollutants. Biochemical adaptation to animal predation and biochemical interactions between plants will be the subject matter of later chapters in this book.

II The Biochemical Bases of Adaptation to Climate

A General

Anatomical and morphological adaptation of plants to different climatic factors is well known and indeed its study is a major part of the science of plant ecology. Everyone is familiar with the ways that desert cacti and succulents are adapted to their parched habitats and are able to reduce moisture loss under the scorching desert sun. This is done by extending the area of soil for water uptake, by reduction of water loss through the leaf or by increased water storage in the tissues. Situations where biochemical features are involved in climatic adaptation are less often considered or discussed. Nevertheless, there is a growing awareness of the need to explore biochemical aspects, from the practical viewpoint.

In recent years, there has been much study of the hormonal control, through the sesquiterpenoid abscisic acid, of moisture loss from plants by stomatal closure. There is practical incentive here in the need to develop drought-resistant crop varieties for growing in marginal desert areas of the world. In Israel, for example, plant scientists are working on the development of agricultural crops which will grow successfully in areas of the Negev desert. Conversely, plants may have to adapt to excess moisture and something is now known of the adaptation of intermediary metabolism to the flooding of plant roots. Also, plants growing in frost conditions undergo biochemical changes in their sap constituents. Perhaps the most dramatic example of long-term biochemical adaptation to climate discovered in recent years is of the special photosynthetic pathway that tropical plants appear to have developed in response to hot and arid conditions. All these topics will be discussed in more detail in the following sections.

B Photosynthesis in Tropical Plants

It has been apparent since the experiments of Warburg (1920) on O2 inhibition of photosynthesis that temperate plants, when subjected to high temperatures (as on a hot summer’s day), do not show the expected increase in photosynthetic rate with temperature that theoretically should occur. Their efficiency in incorporating atmospheric CO2 into respiratory sugar is limited by carbon loss from the well-known Calvin carbon cycle via ribulose bisphosphate and glycollate (see Fig. 1.1) so that a proportion of the CO2 originally absorbed by the plant is lost by ‘photorespiration’ at the leaf surface. Such losses in CO2 conversion to sugar, due to O2 inhibition, are not too serious since high temperatures are relatively infrequent in temperate latitudes. Such losses could, however, be much more considerable in plants growing in the tropics.

Figure 1.1 The Hatch-Slack modification of the carbon pathway of photosynthesis

Evidence that tropical plants such as sugar-cane are able to resist inhibition by high partial pressures of O2 and are able to photosynthesize efficiently in hot climates has only become available quite recently. These new discoveries have shown that such plants differ biochemically from temperate species (Hatch and Slack, 1970; Bjorkman and Berry, 1973). When leaves of sugar-cane are exposed to ¹⁴CO2 for a few seconds, the first compounds labelled are not those of the Calvin cycle but are C4 acids. Such plants have a modified pathway of carbon, which includes a new cyclic system, called the Hatch–Slack pathway, which transports CO2 from the leaf surface to the Calvin cycle (Coombs, 1971). Plants with this so-called Hatch–Slack pathway in effect collect the CO2 which would otherwise be lost by ‘photorespiration’ and ‘drive it back’ into the Calvin cycle to be converted to sucrose (Fig. 1.1). Such tropical plants are called C4 plants (after the four-carbon acids involved in the Hatch-Slack pathway) to distinguish them from the more usual C3 plants which only have the simple Calvin cycle.

This modification in biochemistry is correlated with anatomical differences; plants with the C4 pathway have special mesophyll cells, in which the pathway is located, beside the bundle sheath cells, where the Calvin cycle operates. The anatomical differentiation of C4 plants was recognized long before their distinctive biochemistry was elucidated, the anatomical features of such tropical species being described as the Kranz syndrome. Besides recognizing C4 plants by anatomical observations, it is possible to identify them by determining the relative uptakes of ¹³CO2 and ¹²CO2 by these plants. This ratio is measured in the carbon fixed by the plant as sucrose and can even be determined in dead tissue, i.e. in herbarium specimens. The relative ¹³C/¹²C ratio in C4 plants is between −9 and − 18‰, while in C3 plants it lies between −21 and −38‰ (Gibbs and Latzko, 1979).

The Hatch–Slack pathway was first recognized in sugar- cane, Saccharum officinarum, a member of the grass family, Gramineae. Subsequent investigations have shown that the majority of tropical and subtropical grasses in the sub-families Eragrostoideae, Panicoideae and Arundinoideae have this pathway (Brown, 1975). The ability to achieve optimal photosynthesis under tropical conditions is by no means restricted to grasses and at least ten other families with tropical members contain it. They include the Cyperaceae, Compositae, Euphorbiaceae, Zygophyllaceae and several families of the Centrospermae. In Cyperaceae, the distribution of C4 plants is correlated with taxonomy; the character occurs exclusively in the tribes Cypereae and Fimbristylideae of the Cyperoideae (Raynal, 1973). In general, plants with the C4 pathway are exclusively herbaceous and it is fairly clear that it is an advanced evolutionary condition compared to C3 plants.

An outline of the path of carbon in plants which are adapted to tropical climates is shown in Fig. 1.1. Essentially, the purpose of the Hatch-Slack pathway is to transport CO2 from the outside surface of the leaf to the actual site of photosynthesis in the inner chloroplast. The CO2 is first combined with phosphoenol pyruvate (PEP) (Fig. 1.2) to give oxalacetate which is then reduced to malate, which in turn is decarboxylated to pyruvate. The CO2 released at this stage enters the Calvin cycle by combining with ribulose bisphosphate to give phosphoglyceric acid, the first C3 organic compound of the Calvin pathway. Most of the enzymes required in the C4 pathway are already present in all plant species, the only really distinctive enzyme being pyruvate, Pi dikinase which regenerates PEP from pyruvate to complete the cycle (Fig. 1.2). However, there is evidence that other forms (or isozymes) of the enzymes common to both C3 and C4 plants are required to make the C4 pathway function most efficiently. Equally important for the success of C4 plants is the rearrangement of the cells and membranes which allow the translocation of C4 acids and of pyruvate between the two types of cells.

Figure 1.2 Biochemistry of the C4 pathway in sugar-cane

) to the Calvin cycle can vary in enzymology. Three different enzymes have been recognized (Table 1.2) and C4 plants fall into one or other of three subgroups according to which decarboxylating enzyme is present in the bundle sheath. The scheme of Fig. 1.2 is only strictly followed in plants with the NADP+ malic enzyme; the other two types differ in the actual substrates which are moved to and from the bundle sheath and mesophyll cells (Table 1.2). There are also differences in the organellar localization of the three enzymes within the bundle sheath cell, with the NADP+ malic enzyme operating in the chloroplast, the NAD+ malic enzyme in the mitochondrion and the PEP carboxykinase operating in the cytoplasm with oxalacetate instead of malate as substrate. Plants with the three different decarboxylases can be recognized anatomically as well as biochemically. All three subgroups of C4 plants are found within the grasses, the distribution of the subgroups generally following taxonomic classification.

Table 1.2

Biochemical variations in the C4 pathway of photosynthesis

aFormed from oxalacetate by transamination before transport and yields oxalacetate by deamination before decarboxylation.

All available evidence indicates that species with C4 photosynthesis have evolved from those with only the C3 pathway; if this is true, then one would expect to find intermediate species in nature. The existence of C3 and C4 species within the same genus, as occurs in Atriplex (Chenopodiaceae), suggested to scientists that intermediates might be obtainable by simple cross-breeding experiments. However, although true hybrids were obtainable, they were found to only function photosynthetically as C3 plants in spite of the presence of some C4 characteristics. Continuing search among subtropical plant species has revealed several natural C3–C4 intermediates, the one most extensively studied being the grass Panicum milioides (Rathnam and Chollet, 1980). Such plants are anatomically distinctive from both C3 and C4 types and they have a C4 mechanism which is operational but not as efficient as that in a true C4 species. Finally, the view that C4 evolved from C3 is supported by experiments where it is possible to demonstrate the changeover from C3 to C4 photosynthesis in different leaves on the same C4 plant, as for example in Zea mays (Crespo et al., 1979).

One other point may be made about C4 plants: it is possible that adaptation to retain photosynthetic efficiency in the tropics may have additional benefit in providing resistance to herbivores. There is evidence that herbivores, and particularly grasshoppers, avoid eating C4 plants if given a free choice between both C3 and C4 plants (Caswell et al., 1973; Heidorn and Joern, 1984). The reason for this may be simply due to the anatomical modifications reflected in the Kranz syndrome. Thus, the starch in C4 plants is located further away from the leaf surface and hence is less accessible to feeders. The bundle sheaths of C4 plants also seem to be relatively tough and in grasses at least lignin content seems to be much higher in C4 than in C3 plants. Finally, there is evidence that the availability of nitrogen in C4 plants is less than that in C3 plants. However, not all insects are affected by such factors in C4 plants and, indeed, the lepidopteran Paratryone melane thrives equally well, irrespective of whether it is fed on a C3 or a C4 grass (Barbehenn and Bernays, 1992).

While the main environmental stress suffered by C4 plants is high day temperatures, many plants growing in the drier parts of the tropics and subtropics may also be subjected to drought. This is true especially of desert plants, which may overcome this stress by adopting a succulent habit and storing the water that is available (see also p. 15). Many succulent plants (e.g. Kalanchoe daigremontiana) have been known for a long time to be idiosyncratic in their biochemistry in their penchant for storing organic acid, particularly malic, in the leaves during the night only to dissipate this acidity during the following day. Plants with this unusual behaviour are particularly common among members of the Crassulaceae and the phenomenon has become known as Crassulacean Acid Metabolism (or CAM for short). Recent research (Kluge and Ting, 1978) has now revealed that CAM plants are in fact a special type of C4 plant in which the Hatch-Slack and Calvin cycles operate at separate times over any given 24-h period. Furthermore, CAM plants represent a unique example of a biochemical adaptation in photosynthesis which is linked to the conservation of moisture in an arid habitat.

The pathway of carbon in CAM plants, outlined in Fig. 1.3, is determined by the opening and closing of the leaf stomata. Thus, the stomata remain open at night, when water loss by transpiration is minimal, and CO2 is taken in and combined with PEP to yield oxalacetate and then malate. This latter acid is then stored in vacuolar form until the morning. Soon after dawn, the plant then closes the stomata in order to avoid water transpiration during the heat of the day. At the same time, the second carbon cycle comes into operation and the storage malate provides the main source of CO2 for operating the Calvin cycle, by which sugar is synthesized and then stored. Thus CAM plants are functionally similar to C4 species, except that in CAM the sequential carboxylations of PEP and ribulose bisphosphate are separated in time, diurnally, whereas in C4 plants they are separated spatially, i.e. anatomically. In favourable circumstances, CAM plants may exercise the C3 photosynthetic option entirely. This and other evidence shows that CAM is a special adaptation in xerophytic plants to a harsh desert environment.

Figure 1.3 Outline of the photosynthetic pathway in CAM plants

Two subgroups of CAM plants can be distinguished according to the mechanism of decarboxylation of the malate stored overnight in the vacuole. One subgroup utilizes an NADP- or NAD-linked malic enzyme, while the other uses a PEP-carboxykinase (see subgroup variation in C4 plants, Table 1.2). Some plants which exhibit CAM appear to be able to shift from C3 to CAM in response to changing environmental conditions, e.g. the removal of irrigation. How important such shifts are in natural habitats has yet to be determined but the remarkable gymnosperm Welwitschia mirabilis, a native of the Namibian desert of Africa, has been observed on different occasions to show C3 metabolism, CAM and CAM cycling. The latter situation involves fluctuations in organic acid levels but little nocturnal CO2 fixation (Ting, 1985). Overall, the CAM system of photosynthesis seems to be more flexible in its expression than the straightforward C4 pathway.

CAM plants are as widespread in nature as C4 plants and they have been found in members of some 25 families and 109 genera. Both specializations may be present within the same plant family but in different species. For example, in the genus Euphorbia, C4 occurs in species of the North American subtropics while CAM is present in species of the African tropical zones. While these are the only two modifications in the carbon pathway so far known, relatively few plants have been thoroughly investigated biochemically and it is quite possible that other adaptive sequences in the photosynthetic carbon pathways will emerge as the result of future research.

Even within C3 plants there may well be some adaptations of the photosynthetic enzymes to differing climates. Thus the key enzyme, ribulose bisphosphate carboxylase—oxygenase (rubisco), consists of eight large and eight small subunits and appears to have evolved over evolutionary time from a simpler form found in photosynthetic bacteria (Ellis and Gray, 1986). While the amino acid sequences of the large subunits are largely conserved, those of the small subunits are not. Variation in the small subunits could provide the basis of some thermotolerance; C3 plants may well be adapted to growing optimally in their favoured environments through variations in these subunits. Diurnal variation in rubisco activity is now known to be controlled by a natural reversible inhibitor, 2-carboxyarabinitol-1-phosphate (Gutteridge et al., 1986), and again this control system might have adaptive significance.

C Adaptation to Freezing

Many plants and animals have the ability to resist and survive the below-zero temperatures which they may be subjected to during winter months in northern temperate and arctic regions of the world. In insects, there is evidence that freezing tolerance is achieved fairly simply by the synthesis of glycerol (see Fig. 1.4), which acts as an anti-freeze, exactly as ethylene glycol, CH2OH—CH2OH, does in water-cooled car engines. In higher plants, adaptation to freezing conditions seems to be more complex than this (Levitt, 1980). There is little doubt, however, that freezing tolerance is correlated with an increase of sugar content in the cell sap. Experiments show also that the ability to withstand frost can be achieved artificially by infiltrating plants with