Post on 22-Mar-2018
Cyanogenic Glycosides:
The Plants, The Herbivores, The People
by:
Heather Bowden
Biology SMP
Adviser: Dr. Gorton
2nd
Reader: J. Ramcharitar
Due: May 1, 2009
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Abstract: Cyanogenic glycosides are common defense compounds in thousands of
plants. They function as feeding deterrents because of their bitter taste, and they also
release cyanide to poison herbivores. Although cyanogenic glycosides can benefit
humans as natural insecticides, they can be harmful if they are in our food supply. Over
500 million people depend on a highly cyanogenic plant, cassava (Manihot esculenta).
Humans can exploit this system by inserting genes for cyanogenic glycoside synthesis
into crop plants that otherwise require pesticides. Some specialist herbivores can resist
cyanogenic glycosides and study of these organisms provides clues on how we can
develop ways to reduce the danger of ingestion of cyanogenic glycosides. Reduction of
cyanogenic content in cassava through genetic means functions in cassava leaves and
roots, and leaves have more protein and vitamins than roots. Through a combination of
genetic means, simple education, and incorporation of cassava leaves into cassava root
meal, cassava could become a much safer source of carbohydrates, proteins, vitamins,
and minerals.
Introduction:
Cyanogenic glycosides can be found in over 3000 varieties of plants including
barley, cassava, eucalyptus, flax, white clover, cycas, and apple. Cyanogenic plants are
found all over the world because of the effectiveness of cyanogenic glycosides as a
defense mechanism. Cyanogenic glycosides in themselves aren’t toxic; it is only once
they begin to be degraded that they become harmful. A glycoside consists of a glycone
(sugar) attached to an aglycone (non-sugar), and cyanogenic glycosides have a cyanide
group attached to the aglycone. When the plant cells are damaged mechanically or by an
herbivore, cyanogenic glycosides release damaging hydrogen cyanide because cellular
compartmentalization is lost and they are mixed with β-glucosidase and hydroxynitrile
lyase enzymes in plant tissue or in the herbivore's gut (Tegzes et al., 2003). Cyanide
poisoning disrupts the electron transport train which is responsible for ATP synthesis in
the mitochondria. In particular, cyanide affects proper functioning of an herbivore’s heart
and central nervous system (Kulig et al., 1993).
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Many organisms, including humans, are susceptible to cyanogenic glycosides. Of
the cyanogenic plants, cassava is the most important to humans because its roots are the
primary carbohydrate source in many regions of Africa and South America. The toxic
effects of consuming cyanogenic plants like cassava include mental retardation, paralysis,
even death (Banea-May, 2000, Spencer, 1999).
There are specialist organisms, however, that seem to overcome the toxicity of
cyanogenic plants. We, as humans, are attempting to become specialist organisms
ourselves by manipulating cyanogenic plants, particularly cassava, so that they are fit for
human consumption. Agriculturalists, anthropologists, biochemists, biologists,
geneticists, and horticulturists all share this common goal.
Cassava as well as other cyanogenic plants is made edible through appropriate
preparation. The most effective preparations are very time-consuming but have been used
commercially to produce products such as cassava crackers. However, the proper
preparations sometimes aren't practiced in the villages of third-world countries (Zvauya
et al., 2002). The most popular method of preparing cassava is by turning it into flour.
During this process, the cyanogenic glycosides and enzymes are mixed prior to ingestion,
releasing hydrogen cyanide into juice that is drained away or into the air. The danger is
that the flour could still contain high levels of cyanogenic glycosides if not enough
hydrogen cyanide is released (Zyauya et al., 2002).
There are two current strategies to improve cassava for safe consumption. The
first is to study organisms such as colobus monkeys (Burgess & Chapman, 2005) that
have developed a resistance or tolerance to the effects of cyanogenic glycosides. Some
organisms even benefit from digesting plants with cyanogenic glycosides. For example,
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some fungal pathogens are encouraged by increased concentrations (Lieberei, 2007;
Trione, 1960). In addition, some insects gain a nitrogen benefit from metabolizing
cyanogenic glycosides (Engler et al., 2000). If we can understand how organisms resist
and even benefit from cyanogenic glycosides, we might be able to apply that research to
benefit livestock and humans.
The second strategy to improve cassava is by genetically engineering and
breeding it to reduce its cyanogenic potential. The keenest interest seems to be not in
eliminating cyanogenic glycosides, but in enhancing hydroxynitrile lyase content. An
increase in enzyme production would facilitate cyanide release during preparation.
The first chapter presents background on the variety, distribution, and synthesis of
cyanogenic glycosides and the second will explain some cyanogenic plant/herbivore
relationships. In the third chapter I will delve into the pressing issue of making cassava
safe for human consumption. This section will emphasize the acute and chronic effects of
cyanogenic glycosides, as well as the innovative measures being taken to overcome them.
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Chapter 1: Cyanogenic Glycosides and Plants
Cyanogenic Glycoside Synthesis:
Cyanogenic glycosides are found widely distributed in taxonomically
diverse plants, including monocots, dicots, and ferns (Table 1). Familiar examples
include white clover, bitter almond, cassava, sorghum, lotus, and barley. The type of
cyanogenic glycoside(s) in a plant varies depending on species and there are a variety of
biosynthetic pathways. Cyanogenic glycoside synthesis always begins with one or more
specific amino acids (Figure 1). The precursors of cyanogenic glycosides are common
metabolites, but synthesis of cyanogenic glycosides requires specific enzymes. For
example, synthesis in cassava starts with valine and isoleucine, and involves cytochrome
P450 enzymes. Cytochrome P450 enzymes are common in all domains of life
(Zagrobelny et al., 2004). Researchers in Denmark found that cytochrome P450 enzyme
CYP79D1 catalyzed conversion of valine to linamarin and CYP79D2 catalyzed
conversion of isoleucine to lotaustralin (Andersen et al., 2000). Linamarin and
lotaustralin are the cyanogenic glycosides in cassava, as well as many other cyanogenic
plants. White clover (Trifolium repens L.) synthesizes linamarin and lotaustralin from the
same amino acids as cassava (valine and isoleucine), but conversion of valine to
isoleucine is catalyzed by CYP79D15 (Olsen et al., 2008). The “5”is tagged onto the end
of the name distinguishes it from cassava’s CYP79D1. The P450 enzyme responsible for
the second catalytic step has yet to be determined. Generally, all components for
cyanogenic glycosides are already present and require several genes to catalyze
cyanogenic glycoside synthesis.
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Scientific Name Common Name
Taxonomic
Group
Cyanogenic
Compound(s)
Cyanogenic
Compound Location
Commercially
Produced for
Consumption? References
Prunus dulcis Almond Dicot Amygdalin
Roots, leaves,
kernels Y
Sánchez-
Pérez et al., 2008
Prunus serotina Black Cherry Dicot
Amygdalin &
Prunasin
leaves, fruits,
seeds Y
Sánchez-
Pérez et al., 2008;
Fitzgerald, 2008
Sorghum bicolor Sorghum Monocot Dhurrin young shoots Y
Sánchez-
Pérez et al., 2008; Busk
and Moller, 2002
Heteromoles
arbutifolia California Holly Dicot
young fruit,
young leaves, seeds N Tegzes et al., 2003
Manihot
esculenta Crantz Cassava Dicot
Linamarin &
Lotaustralin entire plant Y Andersen et al., 2000
Lotus spp. Lotus Dicot
Linamarin &
Lotaustralin shoots N
Gebrehiwot &
Beuselinck
Hevea brasiliensis Rubber Tree Dicot
Linamarin &
Lotaustralin seeds, leaves N Lieberei, 2007
Lotus japonicus Lotus Dicot
Linamarin,
Lotaustralin, &
rhodiocyanoside A
and D leaves, young plants Y
Morant et al., 2008;
Forslund et al., 2004
Trifolium repens
L., Fabaceae White Clover Dicot
Linamarin &
Lotaustralin leaves N
Olsen et al., 2008;
Richards & Fletcher,
2002
Hordeum vulgare Barley Monocot Epiheterodendrin malt, leaves Y Nielsen et al., 2002
Eucalyptus
cladocalyx Sugar Gum Dicot Prunasin leaves N Gleadow et al., 1998
Passiflora
capsularis Passion Flower Dicot
Linamarin &
Lotaustralin leaves N Amelot et al., 2006
Cycas revoluta
Thunb Sago Cycad Dicot
Cycasin &
Neocycasin entire plant N Chang et al., 2004
Davallia
trichomanoides
Blume
Squirrel's Foot
Fern Pteridophyte
Linamarin,
Lotaustralin, &
Neolinustatin fronds, fiddleheads N Lizotte & Poulton, 1988
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Figure 1. Metabolism, catabolism, and detoxification of cyanogenic glycosides. Figure 1
from Zagrobelny et al., 2004
Lotus (Lotus japonicus) also contains linamarin and lotaustralin and like in
cassava, linamarin comes from valine and lotaustralin comes from isoleucine (Forslund et
al., 2004). To distinguish the P450 enzymes from those in cassava, they are called
CYP79D3 and CYP79D4. The amounts of cyanogenic glycosides produced are
correlated with the amounts of the enzymes CYP79D3 and CYP79D4. The amounts of
these P450 enzymes are positively correlated with the levels of the enzymes' mRNA's,
suggesting that transcription regulates synthesis.
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Sorghum (Sorghum bicolor), like lotus, shows elevated mRNA expression
associated with increased cyanogenic glycoside production (Bak et al., 2000). In
sorghum, the cyanogenic glycoside dhurrin comes from Tyr in several steps. The P450
enzyme CYP9A1 converts Tyr to Z-P-hydroxyphenylacetaldehyde oxime and CYP71E1
converts that into p-hydroxymandelonitrile (Jones et al., 1999; Busk & Moller, 2002).
Lastly, the enzyme UGT85B1 adds a sugar, completing cyanogenic glycoside synthesis
(Hansen et al., 2003).
Cyanogenic Glycosides’ Catabolism:
When plants are damaged mechanically, cyanogenic glycosides are broken down
by the plant enzymes β-glucosidases into α-hydroxynitrile. α-hydroxynitrile lyase (NHL)
catabolizes α-hydroxynitrile into HCN and p-hydroxybenzaldehyde (Figure 1; Bak et al.,
2000). Cyanogenic glycosides can also release HCN in an herbivore’s gut because of
action by the animal’s digestive enzymes. Animal digestion cleaves off the sugar,
yielding the aglycone, which in the herbivore’s digestive system catabolizes further
release to cyanide, poisoning the herbivore.
Locations of Cyanogenic Glycosides:
It is possible for any plant part to contain cyanogenic glycosides, and some plants
have them throughout, but most plants have them in isolated parts, particularly younger
ones (Table 1). An example of a plant with cyanogenic glycosides throughout is cassava,
although the tuberous roots are of particular importance to people. Similarly, the entire
almond tree contains cyanogenic glycosides, but only the seeds are of interest for human
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consumption. All cycad (Cycas revolute) seeds contain cyanogenic glycosides. Other
plants, like the passion vine (Passiflora auriculata) and sugar gum (Eucalyptus
cladocalyx), have cyanogenic leaves (Amelot et al., 2006; Gleadow et al., 1998).
What determines levels and localization of cyanogenic glycosides is complex.
Concentrations, cite of synthesis, and transport all vary. When it occurs, transport occurs
in the phloem, so girdling experiments can reveal whether transport is important. Girdling
by cutting the bark to sever the phloem is often used to determine whether compounds
are synthesized locally or transported (Jorgensen et al., 2005; Sánchez-Pérez et al., 2008).
The phloem is severed between where the compound is thought to be synthesized and
where it accumulates. Sánchez-Pérez et al. (2008) performed girdling experiments on
almond trees by cutting the epidermis and cambium. This group found that amygdalin
and prunasin were not transported but were synthesized within the seed, although at
different times during seed development.
In contrast to almond, cyanogenic glycosides in cassava are transported. Santana
et al. (2002) first suggested is transport in cassava because they found no linamarase
mRNA in the tuberous roots. Jorgensen et al. (2005) knew that cyanogenic glycosides
were produced in the leaf epidermis and hypothesized that they were transported through
the phloem of the stems. They cut parts of the stem and found a buildup of cyanogenic
glycosides immediately above the area that was cut. This buildup implied that the
cyanogenic glycosides were being transported downward until the cut. Unfortunately, the
researchers neglected to then measure cyanogenic content in tuberous roots. However,
cyanogenic glycoside synthesis in the leaves is important because this will not only lead
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to discoveries about the enzymes involved in transport, but also tells us that the leaves
should be a focal point for making cassava acyanogenic.
Cyanogenic Glycosides and Genes:
In biology, polymorphism results when different combinations of alleles can
generate more than one phenotype. The study of polymorphism is important because it
allows scientists to see how different phenotypes perform in various ecological situations,
thus illustrating potential trade-offs between phenotypes. Genetics enable us to
manipulate existing phenotypes and create others, perhaps generating a polymorphism in
a species where there was none.
Polymorphism in cyanogenic potential makes it possible to study genetics of both
cyanogenic glycoside production and β-glucosidase activity. Researchers at Washington
University studied genetics of cyanogenic glycoside production in white clover (Olsen et
al., 2008). They found that the expression of the gene for the initial enzymes in the
pathway for cyanogenic glycoside synthesis is dominant. The genes responsible are Ac
and Li. AcLi results in lotaustralin and linamarase, Acli results in lotaustralin, acLi results
in linamarase only, and acli results in neither linamarase nor lotaustralin (Daday, 1965).
Another polymorphic species is bird’s-foot trefoil (Lotus corniculatus) (Keymer
& Ellis, 1978). Polymorphism is present for production of cyanogenic glycosides as well
as for β-glucosidases (Scriber, 1978).
Almond also has genetic variability. Almonds differ in cyanogenic glycoside
content, but both bitter and sweet almond trees contain cyanogenic glycosides in all plant
parts. The concentration of cyanogenic glycosides in the seeds determines whether an
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almond tree is bitter or sweet. Bitter seeds contain more cyanogenic glycosides and sweet
seeds contain less. Bitterness is a recessive trait with the homozygous dominant alleles
SkSk or the heterozygous alleles Sksk signifying sweetness and the homozygous recessive
alleles sksk for bitterness (Sánchez-Pérez et al., 2008). What exactly sk is or does is
unknown.
Cyanogenic Glycosides and Plant Pathogens:
Cyanogenic glycosides are not always adequate defense against herbivores or
pathogens, and in a few cases they may encourage fungal growth and attack. This
relationship was first studied in flax (Linum usitatissimum) which attracts the common
pathogenic fungi flax wilt (Fusarium oxysporum) (Trione, 1960). However, this study has
not been replicated or reevaluated, although it is commonly cited. .
Another pathogen, South American leaf blight (SALB), is one of the most
damaging diseases to the rubber industry (Lieberei, 2007). SALB is caused by an
ascomycete Microcyclus ulei. All known methods to combat this disease have failed and
although it is still restricted to South America, accidental transport across seas remains a
threat.
One of the most important species of rubber is Hevea brailiensis, which along
with most other species of rubber, is threatened by SALB (Lieberei, 2007). This crop has
yet to be planted on a commercial scale because all attempts usually result in widespread
infestation. Most rubber is obtained by trees growing naturally in and around the
rainforest. Numerous SALB genotypes exist and no rubber tree including Hevea
brailiensis is resistant to all of them.
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Only new Hevea brailiensis tissues, including leaves, fruits, and stems, are
vulnerable to SALB (Lieberei, 2007). One reason SALB is so pathogenic is that it turns
the plants' cyanogenic defense system against the plant itself. The spores attach to the
leaves and the hyphae to penetrate the leaf tissue. When Microcyclus ulei penetrates a
rubber tree’s leaves, it causes mechanical damage which results in loss of cellular
compartmentalization and mixing of cyanogenic glycosides with β-glucosidases, release
HCN and inhibits scopoletin activity (Lieberei, 2007). Since young tissue generally has
higher cyanogenic glycoside content, it is the most susceptible to SALB. When the
excess HCN diffuses through the leaves, it inhibits the buildup and action of scopoletin
(Lieberei et al., 1989). Scopoletin is another plant defense compound that is known to be
effective against M. ulei, although relatively little is known about its mechanism.
The relationship between sorghum and another fungal pathogen, Gloeocercospora
sorghi is better understood. Its pathogenicity that is also directly correlated with
increasing cyanogenic glycoside concentrations (Wang et al., 1999). G. sorghi infects
sorghum by being virtually immune to HCN. It detoxifies HCN by converting it to
formamide with the enzyme cyanide hydratase (CHT).
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Chapter 2: Cyanogenic Glycosides and Animals
Cyanogenic Glycosides as a Defense Mechanism:
Cyanogenic glycosides are examples of anti-herbivory compounds (Levin, 1976).
Cyanogenic glycosides and cyanide are bitter in taste (Nahrstedt, 1985) and the
intermediates (aldehydes, ketones, HCN, thiocyanate, sulfite, and β-cyanoalanine) are all
highly toxic.
The effectiveness of cyanogenic compounds can be examined by comparing the
extent of herbivory on related plants with varying levels of cyanogenic glycosides. This
situation is seen in polymorphic cyanogenic plants, plants engineered with genes for
synthesis of cyanogenic glycosides, and genera with acyanogenic and cyanogenic
species. For example, when researchers transferred the dhurrin pathway from sorghum
into Arabidopsis thaliana, the flea beetle (Phylotreta nemorum), which normally feeds on
Arabidopsis, ate 80% less leaf material (Tattersall, 2001).
Another study by Dritschilo et al. showed that aphids showed no preference
between cyanogenic and acyanogenic white clover throughout the summer (1979).
However, at the end of August and into September, there were over 60% more aphids
found feeding on acyanogenic plants than cyanogenic ones. This is most likely because
autumn is a growth period for the white clover, and a spike in cyanogenesis is common in
the newly formed parts.
Researchers also looked at the feeding preferences of Japanese beetles (Popillia
japonica) on 27 different species of Prunus (Patton et al., 1997). Japanese beetles fed
almost exclusively on acyanogenic species and did not feed at all on Prunus padus,
which had the highest cyanogenic content at 9.2 mmol/kg-1
. They also looked at feeding
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habits of the beetles on manufactured food consisting of cellulose, water, agar, sucrose,
and several toxic plant compounds. Inclusion of the cyanogenic glycoside prunasin
decreased feeding by 50%. It took more prunasin in the artificial food than in leaves to
reduce feeding. The authors speculate that this may be because cyanogenic glycosides
and β-glucosidases are typically found in the leaf epidermis. When the epidermis is
punctured, more cyanogenic glycosides are released initially than would be in the
artificial food source, where the cyanide is more uniformly distributed. In addition, I note
that the artificial food did not contain any enzymes for catalyzing cyanide production.
The deterrent in this situation would be solely based on the bitter taste and HCN
produced by animal enzymes.
Not all herbivores are insects. Keymer & Ellis (1978) looked at the feeding
preferences of the snails Helicella itala and Cochlicella acuta, pests on numerous crops
including citrus and legumes (Barker, 2002). In this study, they took the two mollusks
and analyzed feeding behavior on cyanogenic and acyanogenic bird's-foot trefoil. The
mollusks preferred acyanogenic plants when the two varieties were presented either
simultaneously or sequentially.
Dirzo & Harper (1982) also did experiments with the mollusks (Agriolimax
caruanae, agriolimax reticulatus, Arion ater, and Helix aspersa). They found that the
slugs and snails completely avoided cyanogenic white clover after an initial taste. Similar
results were seen in field studies by monitoring quadrats of a field mostly covered with
white clover.
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Predators of Plants Containing Cyanogenic Glycosides:
Some species have co-evolved with cyanogenic plants, becoming specialists that
can tolerate or detoxify cyanogenic plant material. Study of specialist species for
cyanogenic plants provides useful insight into the evolutionary strategies for overcoming
cyanogenic plant toxicity. For example, many species of Heliconius butterfly lay their
eggs on passionflower, a producer of cyanogenic glycosides. When the larvae hatch, they
feed on the passionflower. The sara longwing butterfly (Heliconius sara) feeds on
passionflower, but the mechanism by which it tolerates the cyanogenic glycosides is not
known (Engler et al., 2000).
We know more about the Red Passion Flower Butterfly (Heliconius erato). It is
highly resistant to cyanogenic glycosides most likely because it inhibits its catabolism to
produce HCN by not breaking down the cyanogenic glycosides (Amelot et al., 2006).This
species also lays its eggs exclusively on the passionflower, suggesting a co-evolutionary
relationship. Larvae of the moth, Spodoptera frugiperda (fall armyworm) are also
resistant to cyanogenic glycosides, but only marginally so. In a comparison of the fall
armyworm and the sara longwing butterfly larvae feeding on passionflower the sara
longwing butterfly ate more and also sought out the more cyanogenic parts of the
passionflower (Amelot et al., 2006). The fall armyworm initially ate significantly less
than the sara longwing butterfly and had a reduced weight compared to control fall
armyworms. After habituation, however, the fall armyworm's feeding increased and then
plateaued, suggesting an enhanced tolerance, and a possibly less advanced co-
evolutionary relationship. The fall armyworm may develop optimal resistance in the
future. The authors speculate that their feeding pattern of tearing off the leaf, chewing it
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up, and rapidly eating it so that oral protease inactivates the plant’s β-glucosidases may
explain their resistance. I do not think this is plausible due to the significant amounts of
HCN in the feces of the fall webworm. I believe a more likely explanation for its
resistance is like that of H. erato.
Larvae of the fall webworm (Hyphantria cunea) collectively attack leaves of
black cherry (Prunus serotina), damaging them quickly and critically (Fitzgerald, 2008).
Attempting to cause the most mechanical damage to the largest area possible in the least
amount of time with numerous larvae may have been a strategy of the moths to maximize
cyanide release into the air prior to ingestion. This doesn't seem to be the case, however,
because the cyanide content was the same in unharmed and harmed leaves. Instead, the
fall webworm detoxifies the cyanogenic glycosides. Its unique foregut has a pH of 12, the
highest of any insect's foregut. β-glucosidases function very poorly at this pH, preventing
cyanide release. The fall webworm’s extremely long foregut (49% of the whole length)
and its slow digestion also contribute to safe consumption of cyanogenic glycosides. This
detoxification process suggests another co-evolutionary relationship between an
herbivore and cyanogenic plants.
Some mammals, such as the red (Piliocolobus tephrosceles) and black-and-white
(Colobus guereza) colobus monkeys also feed on cyanogenic plants. In a field study,
these monkeys were seen feeding on cyanogenic red stinkwood (Prunus africana)
(Burgess & Chapman, 2005). The authors did not study the tree's biochemistry or other
active components, and mused that this plant may have anti-parasitic effects. A future
experiment should involve closer behavioral monitoring of the monkeys to see if they
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exhibited any indication of upset stomach or gastrointestinal discomfort from the red
stinkwood or if they are truly resistant to its cyanogenic glycosides.
One of the most interesting countermeasures to cyanogenic glycosides is seen in
mammals, particularly those with rumen. Sheep will binge on white clover early in the
day for the nutrition and consequently suffer from the effects of the cyanide (Parsons et
al., 1994). But later in the day they feed on ryegrass, which has less nutrition, but isn't
toxic. This feeding pattern demonstrates a mixed feed regimen where temporary illness is
tolerated for the sake of nutrition. Such observations are important for strategically
designing feed regimens for livestock.
Genetic Engineering to Increase Cyanogenic Glycosides and Enhance Plant Defense:
The use of pesticides is detrimental to the environment and to people (Eddleston,
2006). In fact, a survey of about one-hundred farmers in South Africa showed that
although the majority knew pesticides were harmful, they had misconceptions about the
function and safety of pesticides (Rother, 2006). A proposed method of enhancing
defenses of agricultural plants that doesn't involve pesticides is to transfer genes for
enzymes that synthesize cyanogenic glycosides into them. With food crops, there would
be the added stipulation that these crops would be processed properly and/or the
cyanogens would be localized in plant parts that are not consumed.
Recall that in sorghum CYP79A1 and CYP71E1 are the two P450 enzymes
responsible for making the aglycone of cyanogenic glycosides (Bak et al., 2000).
Researchers in Denmark transferred sorghum's genes coding for these two enzymes into
tobacco (Nicotiana tabacum) and Arabidopsis with mixed results (Bak et al., 2000).
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Noteworthy is that their gene transfers were not always successful, yielding plants with
varying concentrations of cyanide-containing intermediates. The authors concluded that
CYP79A1 is required for the intermediates to be converted to the final end product of
cyanogenic glycosides.
In another study, genes for glycosyltransferase UGT85B1 as well as the P450
enzymes CYP9A1 and CYP71E1 were transfered into Arabidopsis (Tattersall et al.,
2001). Recall that UGT85B1 is responsible for the final step in cyanogenic glycoside
synthesis which is attaching the sugar to the p-hydroxymandelonitrile. In this case,
Arabidopsis produced about the same amount of dhurrin as sorghum (4 mg dhurrin per
gram of fresh weight) and no intermediates accumulated. In plants that had more than 4
mg/gfw, seedling development was slightly decreased. These findings are extremely
significant because mature flea beetles (Phyllotreta nemorum) actually avoided feeding
on the A. thaliana, despite never being exposed to a cyanogenic form of it previously.
The gene transfer was thus successful in deterring at least one herbivore.
One would expect that when Arabidopsis is engineered with all three enzymes,
CYP79A1, CYP79E1, and UGT85B1 they would function most like they do in sorghum.
This hypothesis was tested by engineering one set of plants with CYP79A1 (called 1x), a
second with CYP79A1 and CYP71E1 (called 2x), and a third with CYP79A1, CYP71E1,
and UGT85B1 (called 3x) (Kristensen et al., 2005). The 1x plants were morphologically
similar to wild-type plants and accumulated a large number of dhurrin intermediates, but
no dhurrin. The 2x plants also produced intermediates to dhurrin and showed reduced
growth, morphological changes, and reduced fecundity. Furthermore, levels of
sinapolymalate and sinapolyglucose that function as UV photoprotectants were reduced
19
in the 2x. The 3x plants were most successful, having no morphological abnormalities
and the added dhurrin. The results of this experiment demonstrate the importance being
mindful of potential trade-offs when genetically engineering plants.
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Chapter 3: Cyanogenic Glycosides and People
Third world countries depend on cassava root for carbohydrates, even though it
contains cyanogenic glycosides (Banea-May et al., 2000). Cassava, Manihot esculenta, is
a choice plant because of its easy cultivation and tolerance to stress and herbivory
(Okogbenin et al., 2003). It is able to stay underground, unharvested, for extended
periods of time; this is particularly useful during times of famine (Jorgensen et al., 2005).
Once harvested, however, cassava tends to begin to rot rapidly (Buschmann, 2000) and
are typically inedible after a little over a week. However due to its cyanogenic nature, the
toxic effects of consuming poorly prepared cassava have resulted in growth-delay in
children, paralysis, and death (Banea-Mayambu, 2000, Spencer, 1999).
Cyanogenic Glycosides and Cassava:
A bitter cassava plant with higher the cyanogenic content are also more stress-
tolerant. Sweet cassava with less cyanogenic glycosides is also harvested by monkeys.
For these reasons, many poor countries rely on the bitter type. During times of war or
famine, cyanide poisoning becomes more prevalent because added pressures lead to
improper methods of preparation. The biggest obstacle to proper processing technique is
time. There are solutions, but it is difficult to implement them into the every-day
practices of the people who need them most.
The people in the Nampula Provine in Mozambique process their cassava with a
combination of peeling and grating, then either fermenting or sun-drying, and finally
grinding into flour. Whether heap fermenting or sun-drying are used depends in part on
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the season. In Mozambique, heap fermentation was more common in July while sun-
drying was more prevalent in October (Cardoso et al., 1998).
Fermenting (34.4ppm) was significantly more effective than sun-drying
(64.0ppm), resulting in about half as much residual cyanide (Zvauya et al., 2002). Doing
both (44.4) was better than sun-drying, but counter-intuitively, not as effective as
fermentation alone. The researchers used both methods on the most samples, most likely
because they had anticipated that the combination would have the best results. However,
the surprising results may be because samples were most likely not processed for the
standard fermenting time and sun-drying time since the biggest obstacle to proper
processing technique is time; performing both methods for the full amount of time was
unlikely and most likely accounted for the high cyanide content in the presumably twice
processed cassava. Note that in this study, even the most effective preparation method
left 34ppm residual cyanide, more than 3 times higher than the 10ppm limit suggested by
the World Health Organization.
Table 2. Cyanogenic content of flour from different cassava processing techniques as
determined in an experiment by Zvauya et al. (2002).
Method [Cyanide] (ppm)
Fermentation 34.4
Sun-drying 64.0
Both 44.4
Cassava is also a staple crop in Papua New Guinea, where the people use a
unique method of preparation involving an oven, called a mumu, and coconut cream
(Sopade, 2000). In this study, the researcher mimicked the procedures involving four
different types of mumus. A mumu consists of a pit with stones from a nearby river,
topped with logs and then more stones on top of the logs. The different mumus differed in
22
the layering of the stones and logs, the amount of cassava dough, and the heating times.
Cassava roots were peeled, grated, and squeezed, then turned into dough. No coconut
cream or varying amounts of coconut cream were mixed into the dough. Then the dough
was wrapped in banana leaves. Cyanogenic potential was determined after grating,
squeezing, stirring, and heating in the mumus. The grating also provides more surface
area and mechanical damage for the HCN to be released. Squeezing and constant stirring
helped release free cyanide into the water. In all mumus, cyanogenesis was reduced by
53-79%. The heating of the dough in the mumus was successful because although the
mumu got very hot (120-340ºC), the dough only got to 100-110ºC. The author theorizes
that such high temperatures may have helped maximize the catalytic activity of
linamarase, although he notes that linamarase functions best that 55ºC. Heat also
quickens evaporation of hydrogen cyanide. If anything, coconut cream reduced the
effectiveness of the preparation technique, though the difference was not significant
(P=0.058). The author hypothesized that the potential reduced processing efficiency was
because the lipids in the coconut cream may have prevented breakdown of the cyanogens,
reduced starch breakdown, or reduced microbial activity. The author of this study didn't
speculate as to why the local people used coconut cream except as a lipid source, but I
suspect it is because adding lipids to starches makes them not turn stale as quickly
(Walter, 1998). Chronic cyanide poisoning would not be as much of a concern in Papau
New Guinea as in Africa because cassava is only the fourth most important carbohydrate
source after sweet potato, taro and yam (Sopade, 2000).
23
Cyanide Poisoning from Cyanogenic Glycosides:
Cyanide poisoning can be chronic or acute. Chronic cyanide poisoning occurs
when small doses of cyanide are administered over time, resulting in illness. Acute
poisoning occurs when a high dose of cyanide causes permanent injury or death. Chronic
poisoning is far more widespread. Cassava is the primary carbohydrate source for an
estimated 500 million people, but how many of those suffer from chronic cyanide
poisoning is unknown. There are, however, numerous reports on chronic poisoning in
local regions and villages in African countries such as the Democratic Republic of
Congo, Zaire, and Nigeria.
Chronic cyanide poisoning is aggrevated because it produces iodine deficiency. In
the human body, cyanide is detoxified to thiocyanate (Figure 1), which disrupts iodine
usage by the thyroid gland (Dorea, 2004). Specifically, it competitively inhibits the
thyroid sodium-iodide cotransporter (Braverman et al., 2005). Diseases resulting from
cyanide poisoning (and subsequently iodine deficiency) include konzo, goiter, cretinism,
stunted growth, and ankle clonus. The majority of these diseases are seen in African
people in war-torn regions or regions with poor growing seasons, people who rely largely
on cassava as a cheap calorie and carbohydrate source.
Konzo is the most serious disease and is most common in children over 3 years
old and adult, pre-menopausal African women; it is less common among South
Americans and Asians (Ernesto et al., 2002; Spencer, 1999). The best estimate of
occurrence in Africa is that between 1 and 30 people out of 1,000 to have konzo
(Spencer, 1999). Symptoms of konzo include reduced walking ability, followed by
muscle spasms of the legs, leg muscle tightening, and sometimes permanent paralysis of
24
the legs. Konzo does not affect the brain but the physical problems sometimes become
permanent.
Chronic poisoning from cassava has also been related to goiter and cretinism
(Kulig & Ballantyne, 1993). Goiter occurs when an iodine deficiency causes the thyroid
gland to enlarge. Cretinism occurs when an infant's psychological and bodily
development is retarded from iodine deficiency. Infants can also be affected by a mother's
iodine deficiency when breastfeeding (Dorea, 2004). Iodine deficiency most commonly
occurs among mothers who rely on a cassava-rich diet. The thiocyanate is only
transferred through breastmilk in sufficient quantities to influence thyroid function if the
mothers consume insufficient protein. A diet with insufficiently-processed cassava and
low protein is common for many African women.
Chronic cyanide poisoning can result in stunted growth in children 0-3 years of
age. Humans detoxify cyanide to thiocyanate using sulfur amino-acids, like thiosulfate
(Figure 1). The shortage in sulfur amino-acids results in reduced protein synthesis, and
thus stunted growth. Banea-Mayambu et al. (2000) looked at five villages known for
widespread konzo in the Bandundu region. The people in the Bandundu region of
Mozambique tend to take short-cuts in cassava processing, resulting in widespread
konzo. In fact, they often only soak the tuberous roots in water for short periods of time.
The study involves four villages in the north and one in the south and compared weight
and height of village children to a national standard provided by the National Center for
Health Statistics (NCHS). The mean weight-for-age and height-for age was lower for
both populations. The children from the south had an even lower height-for-age than
those from the north. Children in the southern villages not only had significantly stunted
25
growth, but they also had 15 times more thiocyanate in their urine than the children in the
north suggesting chronic exposure to cyanide. Both populations also had less inorganic
sulfate to use for protein synthesis than a control group of Swedish children. However,
the authors consider that children in the southern village may have had stunted growth for
other reasons as well. The northern children had an extra meal at lunch and the southern
children may have been breast-fed longer because they didn’t have enough solid food,
exacerbating the nutrition deficits.
Ankle clonus is involuntary muscle spasm of the foot and it can be caused by
chronic cyanide poisoning. In Mozambique, 8-17% of the children surveyed had ankle
clonus and all children had elevated thiocyanate levels in their urine (Ernesto et al.,
2002). The normal diet of these children is based on cassava, but clearly it is inadequately
processed.
Acute cyanide poisoning cases are considerately less common and are usually
accidental. Most cases of acute poisoning are seen in poor countries, notably the
Philippines. In May of 2006, in the Philippines, 14 people went to the hospital after
ingesting cassava processed in hot water (Sun Star, 2008). In August of 2008, in the
Philippines, a man didn’t have enough money to feed his family so fed his three children
steamed cassava from his backyard. One died and two were hospitalized (Gomez, 2008).
Cases of chronic cyanide poisoning in the US are few. Sometimes, however,
health enthusiasts make smoothies containing the seeds of fruits such as apricot that have
cyanogenic seeds (Kulig & Ballantyne, 1993). Consumption of cyanogenic seeds results
in isolated cases of acute cyanide poisoning. Although the U.S. and the other developed
26
nations do not depend on consumption of cyanogenic plants like cassava, we are using
our resources to develop less toxic strains of cassava that will benefit the world.
Cassava and Genetic Engineering:
It is crucial to search for and provide the people of Africa with information about
how to prepare cassava properly. Anthropological studies on how specific cultures
prepare their cassava provide a foundation for information. But thus far educating the
people responsible for the plants’ preparation has been inadequate or ineffective as
judged by the prevalence of konzo and other cyanide-related diseases. One of the greatest
obstacles is making the preparations economical and practical.
Another approach to reducing toxicity of cassava is a genetic one. However, the
task is challenging because cassava's improvement involves a tradeoff. As the bitterness
of the plant increases, so does the plant’s resistance to herbivores, but the increasing
levels of cyanogenic glycosides are also toxic to people. Reducing cyanogenic glycosides
in cassava would make it better for human consumption, but might render the crop
susceptible to pathogens and herbivory. Nonetheless, researchers are pursuing a variety
of approaches.
A successful experiment resulting in cassava with only 6-40% of the original
cyanogenic glycosides in its leaves and 1% in tuberous roots utilized RNA interference to
reduce gene expression for the P450 enzymes CYP79D1 and CYP79D2 needed for their
production in the leaves, but not in the tuberous roots (Siritunga and Sayre, 2003).
Linamarin was reduced by 60-94% in leaves and by 99% in tuberous roots. It is worth
noting that the tuberous roots had wild-type expression of both cytochrome P450 genes,
27
despite the drastic reductions in cyanogenic glycosides. This is because only eliminating
the genes for the P450 enzymes in the leaves was necessary because the cyanogenic
glycosides get transported to the roots.
Jorgensen et al. used a similar method to eliminate cassava's cytochrome P450
genes (2005). In one group of plants, they affected CYP79D1 only and in the second they
affected genes for both CYP79D1 and CYP79D2. All transgenic lines produced an initial
leaf with a concentration of cyanogenic glycosides of less than 3% of the wild-type. The
tuberous roots had negligible cyanogenic potential.
However, there was large variability in cyanogenic content of the first unfolded
leaves (<25% cyanide of wild-type); some of the plants had wild-type cyanogenic
potential. As the plant grew, the older leaves contained more cyanide and had closer to
wild-type concentrations. In addition, the plants grew extremely poorly and planting the
transgenic seeds produced wild-type plants. Transgenics will not be a practical method to
reduce cyanogenic potential in cassava until researchers can find a way to reduce the
morphological abnormalities. They were unsure as to why the plants grown in nutrient
medium had significantly less cyanogenic potential than the greenhouse in vivo ones; I
believe that this was due to the presence of mutualist fungi in the greenhouse plants that
allowed for greater nitrogen acquisition for cyanogenic glycoside synthesis. It was also
probably more difficult for the plant roots to obtain oxygen in the nutrient medium
because of the gelatin nature of agar. One successful aspect of this experiment was that
girdling experiments confirmed that although tuberous roots can synthesize cyanogenic
glycosides, mainly they are transferred from shoot tips to tuberous roots. This finding
28
demonstrates another way to reduce cyanogenic glycosides in tuberous roots: by
inhibiting the transfer of the cyanogenic glycosides to the tuberous roots.
Eliminating the major genes for cyanogenic glycosides seems attractive and
eliminating those genes doesn't seem to affect any other biochemical aspects of the plant.
Such a high reduction in cyanogenic potential poses a problem, however because
commercial cassava production would then likely require more pesticides, and that would
most likely be just as undesirable as the results of chronic cyanide poisoning.
Also, elimination of cyanogenic glycoside could affect herbivory. Some plants
have both cyanogenic and acyanogenic strains, and both survive. Often the primary
herbivores are repelled by only minute amounts of cyanogenic glycosides. I would,
therefore, advise against total or near complete reduction of the compounds because
remaining cyanogenic glycosides can be removed by processing. Perhaps a less drastic
reduction in cyanogenic glycosides would be more practical.
Another proposed method to reduce the toxicity of cassava is to enhance
hydroxynitrile lyase (HNL) production (Siritunga et al., 2004). HNL is the enzyme
responsible for the second step in cyanide release: the conversion of acetone cyanohydrin
to cyanide (Figure 1). β-glucosidases are prevalent in leaves and tuberous roots, but HNL
is only common in leaves, resulting in a build-up of acetone cyanohydrin in the tuberous
roots. This group's most successful transgenic line carried three copies of the gene for
HNL and had 1/3 as much acetone cyanohydrin in the tuberous roots. Implications of this
work is that transgenic cassava expressing this gene in the roots would be easier to
process allowing more complete HCN release than conventional cassava. .
29
Cyanogenic Glycosides in Cassava Leaves:
Lastly, if methods for reducing cyanogenic glycosides within the leaves prove
feasible on a commercial level, it might make sense to encourage more leaf consumption
to supplement the tuberous root consumption. People in many countries such as Zaire,
Sierra Leone, Tanzania, and Gabon already ingest cassava leaves as part of their regular
diet (Lancaster & Brooks, 1983). The cassava leaves are typically eaten in the form of a
soup, stew, or sauce, with extra ingredients helping to provide adequate nutrition.
Cassava leaves have substantially more nutrition than roots in the forms of protein
(30-40% leaf dry matter), minerals, and vitamins (calcium, zinc, nickel, and potassium)
(Eggum, 1970; Fasuyi, 2005; Montagnac et al., 2009). However, nutritional value of
cassava leaves varies widely depending on growth factors, environmental factors, age of
plant, time of harvest, etc. (Lancaster & Book, 1982). More importantly, leaf harvest
negatively impacts root size and nutrition. It is therefore critical to determine when and
how much of the plant should be harvested at a time (Lancaster & Brooks, 1983; Fasuyi,
2005; Wobeto et al., 2006). Methods of preparation also affect the nutrition of cassava
leaves.
Cassava leaves have an order of magnitude more cyanogenic glycosides than
roots from bitter varieties and most processing methods, especially those involving heat,
reduce the nutritive value (Lancaster & Brooks, 1983). For example, boiling decreases
vitamin C and vitamin B1 concentrations. It is necessary to determine what methods
remove the most cyanogenic glycosides while retaining the nutrition. For example,
boiling after pounding or chopping is the most common preparation process but, adequate
processing methods are critical. The processes of grinding and boiling leaves results in
30
only 15 mg HCN/kg FW while pounding and boiling results in only 4-11.1 mg HCN/kg
FW (Eggum, 1970; Lancaster & Brooks, 1983). The best cassava leaf preparation method
involves shredding, drying for 1-3 hours, cooking, and turning into flour (Lancater &
Brooks, 1983). Cassava leaves processed with this method result in HCN concentrations
of 0.9-4.5 mg/100 g which is well below the threshold dose for acute toxicity of 50-
100mg HCN (Montagnac et al., 2009).
Cassava leaves, like roots, are toxic but roots do not provide enough nutrition
when eaten alone. It is necessary to supplement a cassava root-based diet with a source of
protein. Since cassava leaves are considered a waste product of the cassava plant and
contain adequate nutrition (with the exception of the amino acid methionine), ingestion of
cassava leaves could easily provide the necessary nutrition.
31
Conclusions:
Cyanogenic glycosides are a profound problem in many parts of the world,
mainly developing countries. Although cyanogenic glycosides can be found in thousands
of plants in numerous forms, for humans the most important cyanogenic plant is cassava
because it is a primary food source, mainly for humans in developing countries.
Cassava's high cyanogenic content is a problem that has resulted in disease and death.
However, several future solutions to this world-wide problem have been proposed.
One possible solution is complete genetic deletion of genes for cyanogenic
glycoside synthesis. However, there are several problems with this
method. These plants growth abnormally, the gene deletion isn’t passed on
the next generation, and they lose their anti-herbivory potential. Therefore,
it may be better to partially reduce the amount of cyanogenic glycosides,
leaving enough to maintain anti-herbivory effects. For example, filter
paper dipped in only 0.1% cyanogenic glycosides in a sucrose solution
was enough to deter feeding by the Mexican bean beetle (Epilachna
varivestis) (Nayar & Fraenkel, 1963). Therefore, reducing cassava’s
cyanogenic potential (1-2% linamarin and lotaustralin in leaves) would not
reduce its anti-herbivory effects (Bernays et al., 1977). If cyanogenic
glycoside-deficient cassava became prevalent, it may also have an
unknown negative impact on specialists and organisms.
Another future solution would be to enhance HNL levels in roots. By
doing this, anti-herbivory effects may be maintained, processing time
would be reduced, and no other biochemical processes would be affected.
32
Incorporation of cassava leaves into diet is also an attractive solution
because cassava leaves have enhanced nutrition in the forms of protein,
vitamins, and minerals. Also, manipulating genes for cyanogenic
glycosides’ synthesis affect leaves directly and roots indirectly. So the
leaves have an advantage of being target for cyanogenic glycoside gene
manipulation while reducing cyanogenic glycosides in roots in the
process.
In conclusion, I propose reducing but not eliminating cyanogenic glycoside
production in leaves and tuberous roots and simultaneously enhancing HNL expression in
roots. With less toxic cassava roots and less bitterness, more nutritious leaves
incorporated into every day diet, cyanide poisoning and malnourishment resulting from
iodine deficiency should be significantly reduced in developing countries. These changes
would render leaves as well as roots edible. Once these methods have proven successful
in cassava, we may then begin adapting them in other commercial plant species such as
maize. Ideally, the plants won't taste so bitter either.
33
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