Richard Synge (1970) - Proteins and Poisons in Plants

Professor Kienitz, ladies and gentlemen, first of all a few words about proteins in general. It’s one of the great achievements of the 20th century starting with Emil Fischer and with Hoffmeister. Not only that we’ve obtained complete clarification of the chemical and physical structures of a few proteins. But also it has been shown that proteins over the entire range of living organisms, viruses, microorganisms, fungi, plants and animals are made up from the same 20 species of L- (levo-) amino acids. The first slide please. These are probably familiar to most of you. And we can go on to the second slide which shows some of the slightly more complicated ones in the 20. Well all the apparent exceptions to this rule of the 20 amino acids such as the occurrence in proteins of hydroxyproline or thyroxine have turned out on closer investigation to verify the generality of the rule as they relate to secondary modifications of ordinary amino acids after they’ve been normally incorporated into a normal peptide chain. This second great clarification that there are these 20 amino acids only in the proteins of the entire living world is the combined achievement of 100’s, even 1,000’s of biochemists. Because no individual could publish it as a discovery, people often seem to underrate its importance. In more recent years it has also become fairly clear that these 20 amino acids are strung together into peptide chains, much in the way that a pattern is woven on a jacquard loom, according to a specification which is coded in the sequence of bases in the chain molecules of the nucleic acids. These nucleic acid bases and the specifying code seem like the 20 amino acids to be common to all living organisms. Living organisms are thus seen in chemical terms as an exceedingly compact group. With this new knowledge it’s interesting to revise Emil Fischer’s calculations about the possible isomerism of the proteins. If the average protein molecule has 400 amino acid residues in its peptide chain or chains, then the permutations of sequence which are possible are 20 to the 400th power, roughly 10 to the 520th power. From our knowledge of coding we can calculate from the DNA content of a single nucleus a maximum for the number of different proteins which the organism may contain. For man the amount of DNA is roughly 6 picograms. And this could code for roughly 10 to the 8th different proteins, which I think must be a generous estimate as not all of the DNA necessarily does code for proteins. We can also, being very generous, estimate that throughout geological time and including the present there may have been 10 to the 20th power of species of living organism on the earth. We know that within a species protein structures show many minor variations between individual genetic strains. But we also know that some protein structures can be identical from species to species. So still being generous let’s look at the next slide. We get approximately 10 to the 30th different varieties of protein as having existed on the earth. And 10 to the 520th as the possible number of isomers, so you can see that only that very, very tiny fraction, roughly 10 to the 500th,1 actual protein in 10 to the 490th possible proteins. An incomprehensibly small proportion of the potential polypeptide sequences have ever existed as the proteins of living organisms. Well when we look at those proteins whose structure has been studied in detail and I’m not going to trespass further on Dorothy Hodgkins ground, except to say that each primary valence molecule or molecular aggregate, although it’s thoroughly interpenetrated with a nacreous medium, it has for such a large molecule a remarkably rigid conformational structure which is determined by the secondary valence interactions of the various parts of the molecule. Well now that we know some of these structures they can be examined in detail by the physical chemists who may before too long be able to tell us exactly what determines these very rigid and specific conformations of the peptide chains. But I think already there’s beginning to be the suspicion that only very, very few of the possible permutations of amino acid sequence would give rise to structures capable of the observed rigidity of conformation. And still fewer of these permutations would give rise to structures capable of the required biological activities. In other words when in the course of evolution a biologically useful protein structure has been developed, there’s been a tendency to conserve it or to elaborate with only rather minor modifications. The comparative study of protein structures is already beginning to give us a whole new insight into the course of biological evolution. But for today’s purpose all I want to remark is that living organisms as they have evolved, have only been able to make stepwise minor modifications to protein structures already tried and trusted. And still less have they been able to modify the biosynthetic mechanism by which the peptide chains are produced or to depart from the 5 different purine and pyrimidine bases. And the 20 different amino acids involved in this “jacquard loom”-like mechanism. The most outstanding case of conservatism in protein structure so far observed is the detailed similarity studied by Emil Smith and colleagues of a histone from the green pea and a histone from the thymus gland of the calf. In evolutionary sense of course the green plants and the animals have evolved from recognisable common ancestors close to the unicellular algae and protozoa. Even between living members of these groups it’s sometimes difficult to draw a clear line. Fungi and microorganisms are evolutionary much more remote. Nevertheless, such conservatism over such a long stretch of geological time does deserve our attention. Well I’ve put these considerations before you so that we could see how safe it has been for animals to give up the biosynthetic mechanisms for producing all the chemically more elaborate amino acids. They could be confident that they could rely on plants, not to mention any fellow animals that they prey upon, to continue to produce nutritious protein. The green plants in general have exceedingly simple nutritional requirements and experience little advantage, indeed much unpleasantness from the existence of animals. Apart that is from some assistance in matters of reproduction and dispersal. Plants also suffer much from parasitic microorganisms. Nevertheless, they have not been able to break out from the biological necessity of producing proteins to perform most of their biological functions. The theme I want to pursue in the rest of this lecture is: How do the green plants nevertheless manage so successfully on the whole to prevent themselves from being devoured? As I work in a food research institute and have gastronomic leanings, I must admit to the use and pleasure, which as human beings we have from devouring as wide as possible a variety of plant materials. And also that we need to improve our methods for protecting from pests those plants which we esteem as food. But I’ll try to keep these utilitarian aspects in a subsidiary position to the evolutionary theme. Well, compared with animals plants are at a notable physical disadvantage when faced by predators. They cannot counterattack or run away or hide. However, they often develop powerful physical defences in the form of thorns, prickles or as in many grasses, knife-like siliceous spicules. We should also note that the social arrangements of animal species are usually such that they do not multiple to the extent of pressing too hardly on their food supply. Professor Wynne Edwards in Aberdeen has indicated the wide variety of social conventions by which different animal species may achieve this aim. Plants have evolved side by side with animals and in many cases have reached a sort of evolutionary equilibrium with their predators. But it can be dangerous for a species when new predators invade its traditional territory. And each plant species requires means for keeping the variety of its predators down to a reasonably low level. Many of these means seem to be chemical ones. To the chemist the remarkable distinction between animals and plants is the fantastic variety of chemical substances produced by the plants. At a guess, the number of known chemical substances obtained from plants exceeds that from animals by a factor of 100 and this factor tends to rise with the years as new natural products in plants are continually being discovered. One may distinguish between primary substances common to animals and plants on which much of their functional biochemistry depends and secondary substances peculiar to plants. It’s reasonable to suppose that many of these secondary plant products are, if they aren’t poisonous, at least repulsive or unpleasant for some of the plants’ potential predators. The best studied chemical group of plant poisons is of course the alkaloids. There has been a curious tendency among evolutionists to deny their biological importance. Even to regard them as wantonly bizarre end products of nitrogen excretion or of excessive photosynthesis. These people have never explained why a plant should require to excrete nitrogen compounds as the element is often in short supply. There are stories that particular strains or groups of rabbits can so adapt themselves as to be able to feed on the deadly nightshade. The next slide please. That is atropa belladonna shown in the slide (Could we have it a little darker now?). They can feed on this plant to such an extent that their flesh becomes poisonous to human beings. I have certainly seen rabbits eating the leaves of the deadly nightshade. Nevertheless, that was in a habitat where its lush vegetation contrasted notably with that of most of the other plants which had been grazed out by both sheep and rabbits. It’s hard to imagine that atropine confers no protection whatsoever on the plant possessing it against herbivores. But alkalides are a well worn theme. And I thought it would be more interesting today to discuss some different kinds of poisons, particularly amino acid derivatives and some miscellaneously other substances. I’ll discuss how some of these may contribute to protecting the plant which possesses them. And in a few cases how the plant protects itself from the toxic effects of the substances which it produces. In many cases the proteins of plants can themselves be toxic, this is most clearly demonstrated when they have direct access to the blood stream. In this respect ricin, a protein of the castor bean, next slide please, ricinus communis, I think that’s a rather imaginative drawing from the 18th century of this plant but ricin is one of the most toxic proteins known. The mistletoe plant, viscum album, also contains toxic proteins of low molecular weight, the viscotoxins. Ricin which I have just mentioned is one of the phytohemagglutinins which are also found in leguminous seeds and all of these seem to have adverse effects when they’re taken by mouth. Leguminous seeds also commonly contain proteins which are inhibitors of the digestive proteolytic enzymes. So their proteins only become digestible after these inhibitors have been inactivated by heat. And heat can also inactivate these phytohemagglutinins. So the invention of cooking can be seen to have greatly extended the range of plant materials which could be eaten by man. The next slide please. There you have the cotton plant, gossypium. And cotton seeds have associated with them glands that are rich in the polyphenolic aldehyde gossypol. The next slide please. That’s gossypol. Well, besides being pretty toxic itself, gossypol inactivates the lysine of the protein nutritionally by Schiff base formation with its epsilon amino groups. And special processing techniques for inactivation or removal of gossypol have had to be developed before the valuable protein of cotton seed could be used as a food. Plants are also notable for containing a number of free amino acids and simple derivatives of amino acids which are not at all widely distributed in nature. More of these are being discovered with every year that passes. And many of them have been shown to be toxic. Could I have the next slide please? One of the firsts to be discovered was djengkolic acid, a constituent of the jenkol bean, pithecellobium lobatum which is popular in the East Indies. Well that substance itself tends to crystallise out as sharp needles in the kidneys of those consuming the beans with unpleasant consequences for them. But they go on doing it. The next slide. This shows you the fruit of blighia sapida known in the West Indies as ackee. And this causes intoxications associated with blood sugar in people who persist in consuming it. Can I have the next slide please? That shows at the top the formula of this hypoglycin and below it shows related amino acid which is present in lychee seeds. In this case the pulp of the fruit which is familiar in Chinese restaurants is quite free from the poisonous amino acid which is entirely present in the seed of the lychee. Next slide please. A major free amino acid constituent of the broad bean, vicia faba is dihydroxyphenylalanine which has been known to be a constituent for more than 50 years and for more than 50 years had the familiar nickname of DOPA dioxyphenylalanine of Guggenheim and Torquati. DOPA has recently been used in massive doses for the treatment of Parkinson’s disease. It’s probably a minor normal metabolite in human beings and scarcely toxic. But in view of these massive doses it’s now being said to have aphrodisiac side effects. Already in 1713 Ramazzini had warned nuns against the amorous thoughts engendered by bean eating. And he recalled that for the same reasons St. Gerome had in former times deplored this practice. But if this is the worst that bean eating can do for human beings nevertheless DOPA may have been involved in the bean as a protection against some potential predator still unknown to us. Next slide please. Linatine which you have there is found in linseed meal from flax, linum usitatissimum and apparently acts as an antagonist of vitamin B6. It’s notable for containing one of the few D amino acids so far found in plants, the proline-moiety there is diproline. And it’s also notable for being a hydrocine derivative. Next slide please. Another amino acid which you have on the left there is azetidine carboxylic acid which is found in the lily of the valley convallaria majalis. This is evidently toxic because the enzymic systems for protein biosynthesis are incapable of distinguishing it from the normal protein constituent proline which you have on the right there. So they incorporate it at random into the proteins being synthesised. Leslie Fowden and his colleagues have shown that the lily of the valley avoids poisoning itself in this way because it has a uniquely specific prolyl transfer RNA synthetase which unlike the corresponding enzyme in other plants and animals distinguishes absolutely between proline and azetidine carboxylic acid. Well now I mention another amino acid, next slide please, indospecine, that’s a tropical forage legume in which it occurs called indigofera spicata. And that’s an Australian cigarette box to give scale I understand. It’s probably toxic by mimicking arginine. And the next slide please. Next slide shows the jack bean canavalia ensiformis which contains canavanine which mimics argenine in a somewhat different way. The next slide please. That shows you the similarity in the formula of those three different amino acids. The indospecine and the canavanine are both definitely toxic. But I don’t think it’s established whether they incorporate it into proteins or whether they interfere in the urea production, the cycle of urea production. Well in districts where the soil is rich in selenium compounds, selenium tends to replace sulphur in the sulphur containing amino acids cysteine and methionine. And the incorporation of these analogues into the proteins of plants or animals takes place and has toxic consequences for them. However a few plants are know which not only flourish on seleniferous soils but actually concentrate selenium compounds in their tissues. One of these, astragalus pectinatus, has been shown to incorporate selenium into Se-methylselenocysteine. Could I have the next slide please? And this astragalus, this legume, unlike normal plants placed in such an environment rich in selenium manages to keep its cysteine, its methionine and its glutathione quite free from selenium analogues. Thus at one stroke such plants divert the toxic effects of selenium from themselves while accumulating substances rich in selenium and powerfully toxic to animals. It was almost certainly plants from this group that in 1295 poisoned Marco Polo’s horses at one of their resting places in western China. In a somewhat similar way fluorine is accumulated by certain plants. Can I have the next slide please? One of these, it’s accumulated in the form of fluoroacetic acid, noticeably in South African plant dichapetalum. This discovery by J.S.C. Marais which he published in a South African veterinary journal in 1944 caused consternation in military circles because fluoroacetic acid had by then been developed as a secret weapon for chemical warfare. In subsequent years R.A. Peters and colleagues showed how fluoroacetic acid acts by producing metabolites which block enzymes of the Krebs tricarboxylic acid cycle. While it’s uniformally toxic to animals, fluoroacetic acid has little obvious toxicity to plants and has therefore found use as a systemic insecticide for ornamental plants. However it’s such a dangerous poison to have in the home that its use has been banned in many countries. From the point of view of plant physiology I think we need to know a lot more about what role if any the tricarboxylic acid cycle plays in the metabolism of green plants. Now the seeds of the sweet pea, next slide please, and of several other legumes have long been known to be toxic. That is lathyrus odoratus, again rather old-fashioned form, one group of toxic substances present which characteristically give rise to a degenerate state of the connective tissues known as osteolaterism is a series of night trials related to simply amino acids. Next slide please. These have been shown to act by interfering with the crosslinking reactions that take lace during the biosynthesis of collagen and perhaps also the connective tissue protein elastin. Another group of substances found in lathyrus species are responsible for a neurotoxic condition known as neurolathyrism. Can I have the next slide please. In particular lathyrus sativus produces such effects through the compound oxalyldiaminopropionic acid. This plant is very drought-resisting and its seeds take a major place in human diets during famine conditions in central India. This toxic substance is water-soluble and it can be largely eliminated by rejecting water in which the seeds have been cooked. There is another advantage that comes from cooking. Well, from the examples which I have given you, you can see that leguminosae have specialised in the use of amino acid derivatives as toxic agents. Perhaps their ability to fix atmospheric nitrogen implies that they can afford to use nitrogen compounds for self-protective purposes more freely than can other groups. Many years ago Stoll and Seebeck showed that allicin, which is a substance responsible for the characteristic odour of garlic, allium sativum, is formed enzymatically from an amino acid precursor alliin. Can I have the next slide please. This happens when the tissues of the plant are crushed. Allicin has definite bactericidal qualities. And these may have contributed to the traditional esteem of garlic as a prophylactic against infectious diseases. My last example involves not an amino acid but agmatine, the decarboxylation product of arginine. Could I have the next slide please? Stoessl has found the most interesting antifungal substance which he has called hordatine in barley. He has shown this to be formed from 2 molecules of p-coumaroylagmatine which you see on the left by oxidative dimerisation and glycosylation. This sort of oxidative coupling of C6-C3 compounds has been postulated as a stage in lignan biosynthesis. But as far as I know, this is the first time that such a dimer has actually been isolated from a plant. And it’s interesting that it should have a biological activity in its own right. Well those are quite enough anecdotes about amino acids and I’ll pass on to the great mass of aromatic and cyclic organic compounds which are accumulated in the tissues of plants: Polyphenols, flavonoids, cinnamic esters, coumarins, tannins, lignans, terpenoids and so on. By sheer bulk these may often confer indigestibility or resistance to microbial attack. There’s a popular tendency to regard plants as largely producers of carbohydrates but there are many plants in which these aromatic or cyclic substances bulk larger than any other class of constituent. Thus, it’s not uncommon for as much as 45% of the dry matter of tealeaves to consist of polyphenolic material. Next slide please. There you have the tea plant, camellia sinensis and my colleague Doctor Tubbs who was formerly with the tea research association in Assam tells me that the tea is an unusually healthy plant which has very, very few diseases. Those things in tea are loosely described as tannin. When you look at the amazing chemical diversity found in these compounds which is fully equal to that found among the alkaloids, and when you consider that each compound involves a highly specific series of enzymes, devoted to its biosynthesis, it seems more reasonable to suppose that these secondary plant products have biological functions shaped by evolutionary processes as yet unknown to us, rather than to regard them as more or less random products of some exuberance of photosynthesis or excretion. Of course, not all of these secondary plant products have the function of being poisonous or disagreeable to potential predators. We seem to be only at the beginning of the discovery of a whole range of chemically varied substances which have hormonal functions within the plant. Moreover, flowers and fruits have to be made attractive to various animal species with a view to fertilisation and dispersal. Chemical means have been employed for the development of colours, flavours and odours. A few cases are established and more should be suspected where plants release chemical substances into air or soil to antagonise the growth of other plants in the immediate neighbourhood. Or the germination of the seeds of other plants or even of their own seeds where physical conditions demand wide spacing. We’re also beginning to realise that these massively accumulated plant constituents play a big part in the formation and maintenance of the soil in which the individual plant grows. The aromatic plant constituents are being seen more and more clearly to be the main precursors of the organic constituents of soil. In dying leaves, polyphenols become oxidised to quinones and these polymerise oxidatively to give large and stable polycyclic condensed aromatic aggregates. Sometimes they’re called humic acid and so on. Could I have the next slide please? During the polymerisation protein and other compounds of nitrogen and sulphur can become coupled to the aromatic aggregates. And this slide shows just one way in which amino compounds are so coupled. These humic substances form an important reserve of nitrogen and sulphur capable only of slow mineralisation and mobilisation. Moreover, the humic substances have important properties concerned with retention of water, maintenance of soil structure, retention of nutrient ions by ion exchange and chelation of trace elements. They may also have more specific biological interactions with microorganisms and with other plants. Could I have the next slide please? It seems fairly sure that in that which is a picture of a beach forest, the absence of vegetation from the floor of the beach forest is not just due to the lack of light. You could well imagine that the beach in its leaves or in its roots produces substances toxic to other plants. Lights please. Organic chemists have discovered and characterised chemically a vast number of substances naturally occurring as secondary plant constituents. In the past this was done at the average effort of about one PhD thesis per substance characterised. And those substances so far recognised can only be a tiny minority of those actually present. But recent improvements in techniques have fantastically reduced the effort required for the chemist to isolate and establish the structure of a plant’s constituents. Particularly relevant in recent years have been the progress in various purification techniques in organic mass spectrometry, in physical methods such as nuclear magnetic resonance and in crystallographic structure determinations. Natural product chemists are on the road to compiling a “Handbuch der Phytochemie” which may before too long include more substances than there will be in Beilsteins Ergänzungswerk is brought up to 1950 which I believe is where Beilstein is proposing to leave matters. Remember that there are a quarter of a million known species of flowering plants alone. What we chiefly need at present is that biologists should take more seriously the functions for which this galaxy of organic compounds has been evolved. Of course in overall energy terms animals, fungi and parasitic and saprophytic microorganisms are largely dependent on green plants for their existence. Yet it’s only when in biological terms much more profound interpretations can be offered for the origin and function of these variegated secondary substances that we shall understand in detail how plants succeed in maintaining themselves so well and in such fantastic variety whilst surrounded by so many potential enemies. As Count Bernadotte mentioned in his opening address, the pesticides now being used by farmers to protect their crops are very dangerous for many kinds of living creatures including ourselves. Intensification of study along the lines I’ve been urging in this lecture may help us to develop pesticides that are less of a general menace. And better still by deliberate analysis of the genetic factors in plants which determine their resistance to pests, we may hope to breed new plant varieties that will not require any external applications of pesticides. Thank you for listening.

Richard Synge (1970)

Proteins and Poisons in Plants

Richard Synge (1970)

Proteins and Poisons in Plants

Comment

The 1952 Nobel Prize in Chemistry was awarded to two British chemists: Archer Martin and Richard Synge. They had jointly discovered partition chromatography, a technique for separating compounds based on the partition between a liquid stationary phase, which is immobilized by attachment to a solid carrier, and a liquid or gaseous mobile phase. This was a ground-breaking contribution, which led to the development of important modern analytical techniques like gas chromatography.Synge’s efforts with respect to partition chromatography stemmed from his interest in amino acids and proteins. Consequently, he begins the present lecture with a few general remarks on these compound classes, but then takes a rather sharp turn towards the fields of plant toxins. He mentions that, since plants cannot attack, run away or hide, there has been evolutionary pressure to develop chemical means of keeping the number of predators down. This pressure, Synge says, has led to a fantastic variety of plant toxins, a “galaxy of organic compounds”. Some of its more prominent representatives are the alkaloids caffeine, morphine, cocaine and nicotine, for example. Synge then embarks on an interesting journey through some particular examples of plant toxins, beginning with those which belong to the group of proteins. A very well-known representative of this group is ricin, which is found in the castor oil plant. Ricin, one of the most potent toxins known, inhibits protein synthesis and causes death by paralysis of vital organs. While, like all proteins, ricin has to be made from valuable nutrients, other plants have found a more economical way of becoming toxic: the accumulation of toxic elements like selenium and fluorine from soil. Synge mentions the African plant Dichapetalum, which builds up fluoroacetic acid. The latter disrupts the citric acid cycle, halting cellular energy metabolism, leading to altered blood pressure, heart failure and death. Today (2013), the compound is used for mammalian pest control in several countries, particularly in Oceania.Synge eventually also discusses the polyphenols, a class of compounds known for its antioxidant properties. Today, certain polyphenols are marketed as nutritional supplements in the context of anti-ageing. However, polyphenols also play an important role in soil formation and maintenance, as Synge points out. Polyphenols originating from decaying plants are the precursors to humic acids, which represent the main organic constituents of soil. There, they form important reservoirs of nitrogen and sulfur and retain water as well as certain metals, Synge explains. In closing his talk, he gives some insight into the discovery processes of natural products like plant toxins, mentioning that until a few years before his talk, the isolation and characterization of a single natural product required the work equivalent of an entire PhD thesis (usually between 3 to 5 years). With respect to emerging analytical techniques like organic mass spectrometry and x-ray crystallography, Synge expected a significant reduction of this timeframe. And he was not mistaken. Since his talk a range of new developments have been made and a significant boost in the rate of natural product discovery could be achieved. Some of these developments, i.e. nuclear magnetic resonance spectroscopy and the electrospray ionization technique, led to the 2002 Nobel Prizes in Chemistry, which was awarded “for the development of methods for identification and structure analyses of biological macromolecules". That such developments were badly needed is illustrated by the extreme case of bombykol, the sex pheromone of the female silk moth. It took its discoverer, 1939 Chemistry Laureate Adolf Butenandt, more than 20 years to isolate and characterize the compound. The complete story can be heard in his 1960 Lindau lecture (LINK).David Siegel

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