Cyanogenic Glycosides: The Plants, The … Scientific Name Common Name Taxonomic Group Cyanogenic Compound(s) Cyanogenic Compound Location Commercially Produced for Consumption? References

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

21

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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