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Pure Appl. Chem., Vol. 73, No. 8, pp. 1325–1330, 2001.
© 2001 IUPAC
1325
Improving self-defense in plants. Martial arts for
vegetables*
Patrick Moyna1,† and Horacio Heinzen2
1Instituto de Biotecnologia, Universidad de Caxias do Sul, Caxias do Sul, RS,
Brazil; 2Facultad de Química, Avda. General Flores 2124, Montevideo, Uruguay
Abstract: From the dawn of agriculture there has been an ever-intensifying human effort to
improve yields by having crops with enhanced biological similarity (i.e., characteristics of
product, maturation time, height, color, etc.). The ultimate stage is to plant a crop where all
individuals behave in exactly the same way, being clones of each other. This very intensive
approach leads to loss of intrapopulation biodiversity and to unstable systems, prone to dis-
astrous losses should anything go wrong.
Biological evolutionary success is usually derived from high adaptability to ever-
changing external conditions. Highly specialized plants (such as certain orchids) or animals
survive by correctly performing a high-wire act of enormous risk. External disbalances have
catastrophic results on these species. Nature excels and corrects imbalances increased biodi-
versity within natural populations. Given this situation, we should study the defensive sys-
tems used by plants and improve on those natural systems.
INTRODUCTION
What we would like to present is a description of our original research ideas, some of the chemical
results we obtained, and how we slowly had to adapt those ideas and our chemical project to what we
learned from biology. Finally, if you allow me, I will suggest some lines which I think we chemists will
have to follow in the future in dealing with agricultural problems.
Like many other postdoctoral students returning to their home countries, we had to start with very
crude laboratory and equipment facilities, and deal with an almost nonexistent supply system. We soon
realized that we would have to tackle a topic that would yield results to very simple chemistry. Even
within phytochemistry we had to opt for an area that included simple solvents and reagents but still
might result in publications.
In the early 1970s desertification in sub-Saharan Africa and other parts of the world was seen as
a very important problem. It was a basic research topic in many areas, evetually leading to the UN
Convention to Combat Desertification, the text of which was approved in 1992 [1]. In South America,
the situation is not so dire, but it was one worth working on. Uruguay and Rio Grande do Sul are in a
region that has reasonable average rainfalls, but alternating periods of intense rain with others of
drought. This has resulted in a well-adapted xerophytic flora.
Analysis of waxes
Our initial studies aimed at the chemical characteristics that give xerophytic plants their ability to sur-
vive during droughts. An insight might be transferred to crop plants by selection of pre-existing char-
*Lecture presented at the IUPAC CHEMRAWN XIV Conference on Green Chemistry: Toward Environmentally Benign
Processes and Products, Boulder, Colorado, USA, 9–13 June 2001. Other presentations are published in this issue,
pp. 1229–1330.
†Corresponding author
acters in the domesticated varieties, resulting in a succesful drought-adaptation program based on chem-
istry. If we could unravel the mechanisms of resistance we should be able to have our first interesting
proposals. After some false starts, we came to the study of epicuticular wax compositions, which
seemed to be a topic that we could tackle even in our very crude working conditions.
Waxes behave as a water-proofing cover to plants and are responsible both for retaining internal
moisture (drying out) and excluding rainwater (avoidance of excessive leaching of metabolites), pro-
tecting internal cells from disruption and death. Epicuticular waxes are a very primitive character in
land plants, as waxes had to be operational even before vegetation managed to take the step from aquat-
ic to terrestrial [2]. The basic component fractions, which are always present, include hydrophobic com-
pounds derived from the acetate biosynthetic pathway. These compounds include straight-chain and
branched hydrocarbons (odd-numbered carbon chains in the C21–C35 range), esters of fatty acids and
fatty alcohols (both even-numbered, the acids in the C16–C32 range and the alcohols in the C22–C32
range), plus the free fatty alcohols and free fatty acids roughly corresponding to those present as esters.
These basic series show a range of components with a bell-shaped distribution, which is reasonable, as
their action depends more on the overall compositions and properties than on those derived from spe-
cific functions or structures [2].
Our first samples came from Cereus peruvianus, a columnar cacti common to all the southern
parts of South America, and from Discaria longispina, an aphyllous Rhamnaceae that is extremely well
adapted to the dry spells of the Rio de la Plata basin. Our idea was that finding the common features in
two different botanical groups would give us an idea of their adaptation mechanisms. The gross results
are shown in Table 1.
We had made the wrong assumptions. Both plants are well adapted to moisture conservation, one
by having a cuirass of water-proofing agents, the other with a wax rich in compounds that are known
to be rather poor water-proofing constituents, as is the case of the triterpenic hydroxyacids. Our error
became more obvious when collecting more information. The epicuticular waxes from grapes (verita-
ble drops of sweet water), keep the fruits from dehydration with a composition that is made up to 60%
by oleanolic acid [5]. Different wax compositions have been reported depending on the age of a plant’s
leaves and between the upper and lower sides of some leaves [6].
All the while we were facing a growing resistance from our research students in tackling the “bor-
ing” TLC and GC work leading to “Tables of simple hydrocarbons”. It was more interesting to study
the physiological and physicochemical activities of specific wax constituents, or to work on the com-
plex and more intriguing range of constituents usually present in the polar fractions of waxes. These
compounds include sec-alkanols, long-chain aldehydes, -diketones, hydroxy-diketones, di-alcohols,
P. MOYNA AND H. HEINZEN
© 2001 IUPAC, Pure and Applied Chemistry 73, 1325–1330
1326
Table 1 Composition of wax fractions [3].
Cereus wax (0.1% fresh plant)
Hydrocarbons 65%
Esters 10%
Sterols 6%
Free acids 5%
Free alkanols 3%
Discaria wax (0.026% fresh stems) [4]
Hydrocarbons 14%
Esters 18%
Free alkanols 22 % (lupeol 14% taraxerol 3%)
Free acids 11%
Hydroxyacids 37% (ursolic 60 oleanoic 38)
many other triterpenes, phenols, resorcinols, flavonoids, coumarins, sugar esters, even caffeine [2,7–9].
Our work went that way.
Including biology in our chemistry
Some of these minor wax constituents, such as caffeine or xanthotoxin, were almost unexplainable
under our initial very “chemical” biology. Why would a plant make such complex compounds, then
extrude them in the wax if they are poor water-proofing agents? Under a more “biological” viewpoint
they are easier to understand. When by some inborn metabolic or morphological change, a compound
such as xathotoxin is extruded with the wax, a chance ecological advantage for the “deviant” individ-
ual can result, making it resistant to herbivores or pests. This more than compensates for the slight loss
of water-proofing capacity.
Here it began to dawn on us that as chemists we were trying to use the very succesful
analytic/synthetic approach linked to experimental confirmation that is the basis of our work. As
chemists we break down a problem into bits, tackle them piece by piece, and verify the results by fur-
ther experiments. When we come with solutions for all the bits, we put them together, and chemical
problems crumble. It usually works remarkably well. This approach has a drawback when applied to
things natural. This is partly due to the fact that many problems in Nature arise, not from the pieces
themselves, but from the way the pieces interplay. Take the pieces apart, solve each on its own, and
when put together again, the problem seems to have moved to some other part of the line. Sometimes,
you sort one problem, you get two [10]!
This situation arises in agriculture, where we have a “non-natural” activity still directly connect-
ed with the “natural order”. We humans have gone from hunter-gatherers to farmers/stock breeders in
the last 10 000 years. What was done at the time? Plants were selected for their direct nutritional value,
for their postharvest qualities and capacity for prolonged storage, or for their adaptability to monocul-
ture. Even the initial stumbling efforts at domesticating plants and animals were such a success that
human history as we know it is a description of the resulting technical and social progression. And
monoculture in the sense of “crops” was the way to progress.
But agriculture was, and still is, carried out within the natural world. So whatever benefits us
humans, 160-lb. omnivores, is bound to be of use to whatever other herbivores are out there. Imagine
being a 10-mg insect trying to survive. After generations of living in a complex and mixed environment,
where one edible plant can be miles away from the next, you fly into a plantation, where every plant is
useful, and where you have thousands and millions of similar plants growing one foot from each other.
It must be Paradise on Earth!
Mind you, from the human farmer viewpoint, that insects munch and suck is bad enough, but as
Jonathan Swift said 300 years back, Nature has seen to it that “big bugs have little bugs upon their backs
to bite ’em and little bugs have littler bugs, and so ad infinitum”.
Insects are excellent pieces for dissemination of plant viruses. Insects move on their own, select
the appropiate plants, and even inject the viruses in their new breeding grounds. Thus, even insects that,
as Pogo would say [11], munch modestly, can graduate from nuisance to full-blown pests, becoming
veritable Trojan horses! As in Troy, this means war. And since Biblical times, this war has been going
on, sometimes with humans winning, sometimes with bugs bringing down whole nations.
We chemists were recruited into this war some 150 years ago. On the basis of what had been so
effective a method for tackling synthetic dyes or explosives, we went to the lab, found out what killed
insects and started working on those lines. Pelt them with chemical brimstone!! And everybody, from
farmers to bakers, governments, and to the general society were ecstatic!! The first insecticides were
crude and evil-smelling, others were too toxic for safe use. We modified those basic structures, found
some that were more specific to insects. We even went back to Nature to find which compounds were
active and how, and back to the lab to build on those ideas [12]. We improved the lethality, specificity,
permanence, degradability, and their ability to stick or unstick on plant surfaces.
© 2001 IUPAC, Pure and Applied Chemistry 73, 1325–1330
Improving self-defense in plants 1327
We can check what happens on a plant surface when it is growing exposed to natural conditions
by observing plants in a vineyard left on their own and being colonized by nonpathogenic epiphytic
microorganisms. This is interesting because these saprophytic nonpathogenic microorganisms tend to
cover all the available leaf surface, affecting the wetting of the surface, and in turn are controlled by the
water repellency of the surface [13]. In Table 2 we see that otherwise healthy leaves carry large loads
of saprophytic flora, and cultures of a swab taken on the leaf surface give high counts of colony form-
ing units (CFU) in different growth media. When the leaves are treated with the traditional copper salt
sprays, the CFU counts are strongly depressed. The “normal” flora can be almost exterminated when
more active pesticides are used. When sprayed with dilute essential oils it can be affected in varying
percentages, and these treatments probably are similar to the conditions plants face in the wild (not
under cultivation). With some oils sprays CFU counts are affected but not exterminated [14]. In our
efforts to eliminate “pests” we sometimes exterminate nonpathogenic saprophytes. And when they are
gone we run the risk of seeing the same leaves occupied by nastier neighbors!
Nature, except for sporadic catastrophes, is very parsimoniuous when dealing with the extermi-
nation of species. Each species works because it has an adequate adaptation to a certain ecological
niche. Conditions tend to change gradually in Nature. Sometimes, they move further, sometimes they
go backwards. They are seldom (NEVER) static. From the insects’ point of view, the new natural situ-
ation was the appearance of a shower of noxious and toxic chemicals that killed them.
Plants and animals in Nature are not lonely creatures. They are parts of greater populations that
are connected in space and time. If the external situation changes gradually, as they usually do, within
that connected population there are individuals with intrinsic properties that make them more efficient
(or less inefficient) in the new situation and thus adaptable to the changes. They and their offspring sur-
vive and thrive, the others slowly fade away. When we observe Nature we see a specific set of species
and conditions, but they are not immutable, they are continuosly being selected by external changes.
Some types become extinct, but their relatives survive. Extinction in this way is part of the evolution-
ary process, in the same way death is a part of life. So agrochemicals became the new driving force for
the selection of resistant individuals and for the breeding of “whatever-resistant” insects.
Control of resistance
Are we damned to always select the more resistant pests when we use chemistry to solve our problems
with Nature? Now, a word of hope. The first area in which the impact of using chemicals on natural
populations could be observed was the use of antibiotics. They were supposed to be the end of infec-
tious diseases. Nobody took Darwinian “survival of the fittest” into account. Yes, we did exterminate
innumerable noxious (and not so noxious) microorganisms. But we did it at a pace that was almost nat-
ural in its gradualism, without ever managing complete exterminations. We allowed the fit to survive,
P. MOYNA AND H. HEINZEN
© 2001 IUPAC, Pure and Applied Chemistry 73, 1325–1330
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Table 2 (14) CFUs/g leaf.
Treatment None Cu spray Rosemary Citronella Camphor
Growth
Medium
EMB 10400 360 1100 8300 3220
LB 20500 240 900 7200 2860
WLN 5700 340 2000 10000 3280
YEPD 7000 280 600 5100 3140
PDA 9600 70 500 5000 2980
to go through a mixed population phase, and now we clamor because we have selected populations that
are resistant to our antibiotics of first, second, or whatever generation!! This was a textbook demostra-
tion of Darwinian evolution in action.
Sometime back Darwinian retro-evolution was tried on this field. Macrocyclic antibiotic ill-use
had resulted in microbial populations with more than 20% resistance to these last-resort antibiotics, get-
ting to the point of making them useless. Resistance had increased in all samples taken from throat
swabs of patients in Japan, North America, and Europe. In the early 1990s in Finland it was decided to
apply a program of strict control in their use. No more erythomycin for colds and other virus infections.
Use dropped by 80%. The interesting aspect is that 4–5 years later, resistance started dropping back
from almost 20% to 8%, and was still dropping at the last count. Darwin was right. The selective pres-
sure dissapears, the selection for resistance drops, a balanced population takes over again [15]!
We should be thinking along these same lines when dealing with agriculture, and we chemists
have to learn to think not about exterminating our foes, but of outclassing them.
We have to learn how to work on a regulated biodiversity, to use chemistry to analyze the best
mixes of crop varieties to keep pests and attacking insects always on the verge of their complete adap-
tation to the crop, but stopping them before the adaptation is complete and the attack irresistible. We
can study the plants we use as crops, and taking wheat as an example we soon realize that we have
exploited only a small group of a whole crowd of relatives, lesser members that can be incorporated to
create a biodiverse population that can be economically used. Instead of working to make our mono-
cultures less and less biodiverse, we should consider making them biodiverse, but manageable and
exploitable.
We can add a temporal scale to biodiversity, outmaneuvering our enemies by changing the envi-
ronment before they manage to evolve and carry out one of their massive attacks. By adopting natural
rythms and tactics, we might come with a more sustainable system, keeping our enemies constantly off
balance. The situation is critical, but not hopeless.
How will a small research group from Latin America working on plant epicuticular waxes sur-
vive? We will have to analyze the importance of waxes in the natural system in which they have evolved
and translate that into useful adaptations in a scheme where you try to contain but not exterminate. For,
how do insects recognize the right plants for feeding or oviposition? By smell and taste of the most
external layers, in particular, the epicuticular waxes!! So there still is an interesting prospect for con-
tinuing and improving a study that began because the only solvent we could count on getting was petrol
ether!
ACKNOWLEDGMENTS
Let me thank a long list of colleagues who have put up with me for these many years, especially those
from the Instituto de Biotecnologia at the Universidade de Caxias do Sul (Brazil), and those from the
Pharmacognosy and Natural Products Laboratory at the Facultad de Química, Montevideo (Uruguay),
as well as the funding organisms in both countries that financed our work, FAPERGS in Brazil and
PEDECIBA in Uruguay.
REFERENCES
1. UN Resolutions 44/172 A (19 December 1989) and 44/228 (22 December 1989).
2. G. Bianchi. “Plant waxes”. In: Waxes: Chemistry, Molecular Biology and Functions, R. J.
Hamilton (Ed.), Oily Press, Dundee (1995).
3. J. Hughes, G. Ramos, P. Moyna. J. Nat. Prod. 43, 564–566 (1980).
4. P. Moyna and H. Heinzen. An. Acad. Brasil. Ciênc. 57, 301–303 (1985).
5. F. Radler. Amer. J. Enol. Vitic. 16, 159–163 (1965).
6. S. D. Eigenbrode, M. White, J. L. Tipton. J. Kansas Entomol. Soc. 72, 73–81 (1999).
© 2001 IUPAC, Pure and Applied Chemistry 73, 1325–1330
Improving self-defense in plants 1329
7. S. García, C. García, H. Heinzen, P. Moyna. Phytochemistry 44, 415–418 (1997).
8. E. Wollenweber. Naturwissenschaften 76, 458–463 (1989).
9. M. L. Athayde, G. C. Coelho, E. P. Schenkel. Phytochemistry 55, 853–857 (2000).
10. R. Fortey. Life, Knopf, New York (1998).
11. W. Kelly. The Impollutable Pogo, Simon and Schuster, New York (1970).
12. P. C. Vieira, J. B Fernandes C. C. Andrei. In Farmacognosia, da Planta ao Medicamento 3rd ed.,
C. M. Oliveira, E. P. Schenkel, G. Gosmann, J. C. P. de Mello, L. A. Mentz, P. R. Petrovick (Eds.),
Editoria da Universidade, Porto Alegre (2001).
13. D. Knoll and L. Schreiber. Microb. Ecol. 41, 33–42 (2000).
14. J. Carrau and M. Camassola, personal communication.
15. H. Seppälä, T. Klaukka, J. Vuopio-Varkila, A. Muotiala, H. Helenius, K. Lager, P. Huovinen. N.
Engl. J. Med. 337, 441–446 (1997).
P. MOYNA AND H. HEINZEN
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