Asparagine in Plants

REVIEW ARTICLE

Asparagine in plantsP.J. Lea1, L. Sodek2, M.A.J. Parry3, P.R. Shewry3 & N.G. Halford3

1 Department of Biological Sciences, Lancaster University, Lancaster, UK

2 Departamento de Fisiologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas – SP, Brazil

3 Crop Performance and Improvement Division, Rothamsted Research, Harpenden, UK

Keywords

Accumulation; acrylamide; asparagine; food;

potato; wheat.

Correspondence

P.J. Lea, Department of Biological Sciences,

Lancaster University, Lancaster LA1 4YQ, UK.

Email: [email protected]

Received: 26 October 2006; revised version

accepted: 3 November 2006.

doi:10.1111/j.1744-7348.2006.00104.x

Abstract

Interest in plant asparagine has rapidly taken off over the past 5 years following

the report that acrylamide, a neurotoxin and potential carcinogen, is present in

cooked foods, particularly carbohydrate-rich foods such as wheat and potatoes

which are subjected to roasting, baking or frying at high temperatures. Sub-

sequent studies showed that acrylamide could be formed in foods by the thermal

degradation of free asparagine in the presence of sugars in the Maillard reaction.

In this article, our current knowledge of asparagine in plants and in particular its

occurrence in cereal seeds and potatoes is reviewed and discussed in relation to

acrylamide formation. There is now clear evidence that soluble asparagine accu-

mulates in most if not all plant organs during periods of low rates of protein

synthesis and a plentiful supply of reduced nitrogen. The accumulation of aspar-

agine occurs during normal physiological processes such as seed germination and

nitrogen transport. However, in addition, stress-induced asparagine accumula-

tion can be caused by mineral deficiencies, drought, salt, toxic metals and path-

ogen attack. The properties and gene regulation of the enzymes involved in

asparagine synthesis and breakdown in plants are discussed in detail.

Introduction

Asparagine was the first amino acid to be isolated from

plants, 200 years ago (Vauquelin & Robiquet, 1806),

after the characteristic cubic crystals had been observed

in the concentrated solutions of the sap of Asparagus

species by Delaville (1802). Asparagine is an amino acid

amide that has a molecular mass of 132.12 and an iso-

electric point of 5.41. Although soluble in both acids and

alkalis, asparagine is only moderately soluble in water

and readily forms white monohydrate crystals. Further-

more, asparagine has a N:C ratio of 2:4 (Fig. 1), which

makes it an efficient molecule for the storage and trans-

port of nitrogen in living organisms.

Although it was not initially thought to be a constituent

of proteins, asparagine was shown to be present in the

enzymic digest of the Brazil nut seed storage protein edes-

tin (Damodaran, 1932) and is now known to be present

in most proteins. Although it lacks the negative charge

of the free carboxylic acid group of aspartate, asparagine

retains some polarity and frequently plays a key role in

the active site of enzymes (Mansfield et al., 2006). In

addition, a range of oligosaccharides may be attached to

the amide group of protein-bound asparagine, catalysed

by glycosyltransferases (Lerouge et al., 1998; Bencur et al.,

2005).

The interest in plant asparagine has really taken off over

the past 5 years following the report that the neurotoxin

and potential carcinogen acrylamide was present in

cooked foods, particularly carbohydrate-rich foods such

as wheat and potatoes which are subjected to roasting,

baking or frying at high temperatures (Tareke et al.,

2002). Subsequent studies showed that acrylamide could

be formed in foods by the thermal degradation of free

asparagine in the presence of sugars in the Maillard re-

action (Mottram et al., 2002; Stadler et al., 2002; Zyzak

et al., 2003), leading to a series of studies (described later

in the review) on the levels of asparagine in cereals and

potatoes and their relationship to the levels of acrylam-

ide in baked and fried products. It is therefore appropri-

ate to review our current knowledge of asparagine in

plants, including its distribution, synthesis and degradation,

Annals of Applied Biology ISSN 0003-4746

Ann Appl Biol 150 (2007) 1–26 ª 2006 The Authors

Journal compilation ª 2006 Association of Applied Biologists

1

biological role and relationship to acrylamide formation

during processing.

The occurrence and accumulation of

asparagine in plants

Asparagine has a high ratio of N:C, is a substrate for only

a few enzymatic reactions in the soluble form and has little

net charge under physiological conditions. It therefore

forms an ideal storage compound and accumulates under

a range of situations. Similarly, it is a major transported

form of nitrogen, particularly in leguminous plants.

Transport of reduced nitrogen in annuals

Asparagine is the major transport compound in the xylem

from the root to the leaves and in the phloem from the

leaves to the developing seeds in a range of plants. A con-

siderable amount of information has been obtained on

these transport processes by Pate, Atkins and colleagues,

following their elegant and detailed studies on legumes

(Pate, 1980). In nitrogen-fixing Lupinus albus, asparagine

was the major amino acid in all plant parts and could

account for 60–80% of the total amino acids in nodu-

lated roots, leaves and fruits (Pate et al., 1981). Asparagine

was also the major component of the xylem (45–50%)

and less so of the phloem (20–30%), where glutamine also

made a significant contribution. Interestingly, the pro-

portions remained relatively constant during the 60-day

growing period (Pate et al., 1979; Atkins et al., 1983).

However, if the roots were treated with argon, thus

removing nitrogen and preventing fixation, there were

rapid decreases in the concentrations of amino acids and

in the proportion of asparagine, in both the xylem and

phloem (Pate et al., 1984).

As we have seen above, asparagine is the major end

product of nitrogen fixation in Lupinus species and is

present in high concentrations in the nodules and root

xylem exudates. This is also the case in other temperate

legumes such as Medicago (Snapp & Vance, 1986; Ta et al.,

1986) and Pisum (Fig. 2) (Scharff et al., 2003). However,

the precise pathway by which the ammonia is incorpo-

rated into asparagine within the nodule is still a matter

of debate (Prell & Poole, 2006). Even in peanut (Arachis

hypogea), which is known to contain the very unusual

amide c-methyleneglutamine (Fowden, 1954), aspara-

gine was the major amino acid in the nodules and xylem

exudates of nitrogen-fixing plants (Peoples et al., 1986).

However, not all nitrogen-fixing legumes use asparagine

as the major nitrogen transport compound. Tropical le-

gumes belonging to the Phaseolae tribe, e.g. soybean

(Glycine max) (Rainbird et al., 1984; Herridge & Peoples,

1990) and cowpea (Vigna unguiculata) (Peoples et al.,

1985; Atkins et al., 1988), use the ureides allantoin and

allantoic acid, derived from purine metabolism (Smith &

Atkins, 2002).

Recent data published by do Amarante et al. (2006)

have proved useful in improving our understanding as

to when legumes use asparagine rather than ureides as

nitrogen transport compounds in the xylem. The data

shown in Fig. 3 clearly indicate that ureides are the major

form of N transport compounds in nodulated G. max, V.

unguiculata and Phaseolus aureus, whereas amino acids

and nitrate predominate when the plants are not fixing

nitrogen. In the species that form small or zero amounts

of ureides (Crotalaria juncea, Pisum sativum and L. albus),

only amino acids were found in the xylem sap of nodu-

lated plants, whereas both nitrate and amino acids were

found in the plants not undergoing nitrogen fixation.

A more detailed analysis of the xylem amino acid

fractions of six different plants (do Amarante et al., 2006)

indicated that under most circumstances, asparagine was

still the major form of N transport, although glutamine

was also prominent in the nitrogen-fixing, symbiotic,

Figure 2 The concentration and 15N labelling of amino acids in the

nodules of Pisum sativum following 8 h of incubation in 15N2. Data from

Scharff et al. (2003), with permission. FW, fresh weight.

Figure 1 The structure of asparagine.

Asparagine in plants P.J. Lea et al.

2 Ann Appl Biol 150 (2007) 1–26 ª 2006 The Authors


ureide-forming plants (Fig. 4). The difference in aspara-

gine between symbiotic and non-symbiotic plants was

much less apparent in the non-ureide-forming plants.

The data clearly indicate that even in those plants classi-

fied as ureide formers, a significant quantity of nitrogen

was also exported in the xylem as the amides, glutamine

and asparagine. The importance of the proportion of

asparagine in the xylem as an indicator of the nitrogen

status of a plant can be seen from the data shown in

Fig. 5. Four days of maintaining the plants in nitrogen-

free conditions caused a reduction in the proportion of

asparagine and an increase in aspartate in all the plants

tested, irrespective of whether nitrate or nitrogen fix-

ation was the original source of nitrogen (do Amarante

et al., 2006).

Figure 3 Ureide, amino acid and nitrate concentrations in the xylem

of nitrogen-fixing symbiotic (S) and nitrate-grown non-symbiotic (NS)

versions of six legume species. Data from do Amarante et al. (2006),

with permission.

Figure 4 Amino acid composition of the xylem of nitrogen-fixing sym-

biotic (S) plants, nitrogen-fixing symbiotic plants treated for 4 days with

nitrate (S + NO3) and nitrate-grown, non-symbiotic (NS) plants. Data

from do Amarante et al. (2006), with permission.

Figure 5 Asparagine (shaded) and aspartate (open) content of the

xylem of control nitrogen-replete plants (upper bar) subjected to

nitrogen-free conditions for 4 days (lower bar): (A) non-symbiotic plants

grown on nitrate: (B) nitrogen-fixing symbiotic legumes. Data from do

Amarante et al. (2006), with permission.

P.J. Lea et al. Asparagine in plants



3

In nodulated soybean, such changes in asparagine

transport in the xylem were associated with a decrease

in asparagine synthetase (AS) activity of the nodules

(Lima & Sodek, 2003).

Transport and storage of reduced nitrogen

in perennials

Deciduous trees and other woody plants store reduced

nitrogen in the form of either vegetative storage proteins

or amino acids over the winter period. The nitrogen is

rapidly remobilised in the spring and transported to the

developing leaf and flower buds in the form of soluble

amino acids (Millard, 1996). In addition, there is a mas-

sive recycling of nitrogen following the metabolism of

phenylalanine and tyrosine to form lignin (Canton et al.,

2005). Early work on apple (Malus domesticus) indicated

that asparagine and arginine were the major transport

compounds in the phloem, with the former used for

short distance transport and the latter for long distance

transport (Tromp & Ovaa, 1971). Data obtained by

Malaguti et al. (2001) showed that in apple, asparagine

accounted for more than half of the total amino acid

nitrogen in the xylem, followed by arginine, glutamine

and aspartate, at the time of full bloom. Later in the sea-

son, asparagine still predominated in the xylem but was

derived from remobilisation and also nitrogen taken up

by the roots (Tromp & Ovaa, 1976).

Asparagine often in combination with glutamine and

arginine has been shown to be the major nitrogen trans-

port compound in citrus (Citrus unshiu) (Kato, 1981),

poplar (Populus spp.) (Escher et al., 2004), beech (Fagus

sylvatica) (Nahm et al., 2006) and cherry (Prunus avium)

(Millard et al., 2006). Aidar et al. (2003) studied a Brazil-

ian Atlantic forest community, aiming to characterise

the strategies involved in tree N acquisition and trans-

port during different phases of forest succession. For the

pioneer species, asparagine was the main nitrogen trans-

port compound, comprising nearly 50% of the xylem

nitrogen. The leguminous early secondary species

transported a variety of compounds, but in all tree spe-

cies, asparagine accounted for 24–76% of the xylem

nitrogen.

In contrast, in evergreen conifers, there is evidence that

asparagine does not play a major role in the storage and

transport of nitrogen in the mature tree. In Scots pine

(Pinus sylvestris), arginine and glutamine were the major

forms of soluble nitrogen in the bark, wood and needles

and were increased by N fertilisation (Nordin et al.,

2001). Significant quantities of arginine were lost when

senescent needles were shed, particularly from well-

fertilised trees (Nasholm, 1994). High concentrations of

arginine were also detected in the needles of Norway

spruce (Picea abies), mostly when minerals other than

nitrogen were limiting (Ericsson et al., 1993). Schneider

et al. (1996) carried out a direct comparison of the coni-

fer P. abies and deciduous Fagus sylvatica grown under

field conditions of high nitrogen. The amino acid content

of the leaves, phloem and xylem of the two trees were

determined during the period from April to September.

In P. abies, soluble asparagine was not detected in any

appreciable amount at any time in any tissue through-

out the growing season. In F. sylvatica, asparagine was

the predominant amino acid in the leaves and xylem but

not phloem in April and May only. However, glutamine

and arginine were often present in high concentrations

in both trees.

It is clear, therefore, that asparagine can act as a storage

and transport molecule, often at the same time within the

plant. However, arginine and glutamine are also able to

carry out the same role. There is also evidence of species

differences in the use of these compounds, as well as

temporal and tissue differences.

Senescence

King &O’Donoghue (1995) have examined in some detail

the metabolic processes taking place in harvested spears

(shoot tips) of asparagus (Asparagus officinalis), the original

source of asparagine. Initially, the major soluble amino

acid in the tips was glutamine, but following harvest, the

asparagine concentration increased from 6.5 mg g21 dry

weight (DW) to 28.6 mg g21 in 48 h. Within the 48-h

storage period, the rate of respiration fell dramatically,

and there was an almost complete depletion of soluble

monosaccharides. During the second 24-h period, the

concentration of ammonium also increased (Eason et al.,

1996). It has been proposed that the asparagine content

could be used as a direct ’freshness test’ of asparagus

spears (Hurst et al., 1998), as increases from 50 to more

than 400 lmol g21 DW (Fig. 6) were detected. Large in-

creases in asparagine and glutamine concentrations and

a reduction in soluble sugars were also detected in broc-

coli florets for up to 80 h following harvest (Downs et al.,

1997). The data suggested that following senescence

induced by the

harvesting of either asparagus spears or broccoli florets,

asparagine acts as a store of nitrogen, when the supply

of soluble carbohydrate is severely reduced.

Asparagine has also been shown to be a major product

during metabolism in senescing leaves, although it should

be noted that themajority of experimentswere carried out

on leaves that had been detached from the plant and did

not senesce naturally. Mothes (1926, 1940) showed that

when leaves of Vicia faba and Phaseolus multiflorus were

detached, the protein level decreased with an equivalent




rise in soluble amino acids, in particular glutamine; this

was followed by a rapid increase in asparagine. In

detached leaves of tobacco, work by Vickery et al. (1937)

showed that leaves maintained in the dark formed more

asparagine than leaves maintained in the light, which

synthesised glutamine preferentially. Further work by

Yemm (1950) using excised barley leaves indicated that

a succession of reactions followed protein breakdown,

involving the production of high concentrations of glu-

tamine, followed by asparagine and finally ammonia. A

similar short-term accumulation of a high concentration

of asparagine occurred following the incubation in the

dark of leaves of Lolium temulentum (Thomas, 1978), oats

(Malik, 1982) and wheat (Peeters & Van Laere, 1992).

When detached shoots of P. sativum were incubated in

the light for 72 h, there was little change in the concen-

trations of the major amino acids. However, if the shoots

were incubated in the dark, the asparagine concentra-

tion (but not that of any other amino acid) increased

from 3.17 to 24.0 lmol g21 fresh weight (FW) during

the 72-h period (Joy et al., 1983). Readers who are

molecular geneticists will be pleased to learn that even

Arabidopsis thaliana plants, when placed in the dark,

accumulated high concentrations of asparagine after

6 days (Fig. 7) (Lin & Wu, 2004).

Seed germination

Very early studies showed that asparagine accumulated to

high concentrations in the germinating seedlings of Vicia

sativa and a range of Lupinus species. Table 1 shows data

from the classic experiments of Schulze (1898) with

Lupinus luteus, where it can be seen that the accumulation

of soluble asparagine can account for more than 50% of

the total nitrogen in the seedlings. Subsequent data

showed that the asparagine was distributed throughout

the seedlings, with values (expressed as % of DW) for

cotyledons (3.73), plumules (1.41), stems (4.48) and roots

(2.17). In stems, asparagine accounted for more than

66% of the total nitrogen. Later experiments by Pria-

nischnikov (1922) showed that the addition of ammonia

could stimulate the accumulation of asparagine even

further in a range of germinating seedlings, provided a

source of carbohydrate was available. Further work

showed that as well as in legumes, Papaver somniferum,

Pinus sylvestris and Tropaeolum majus also accumulated

asparagine during germination, while Cucurbita pepo,

Helianthus annuus and Linum usitatissimum accumulated

asparagine and glutamine. The early work on the nitro-

gen metabolism of germinating seedlings has been dis-

cussed in detail by Chibnall (1939) and Mckee (1962).

With the development of accurate and efficient auto-

matic equipment for the analysis of amino acids and

amides, the accumulation of asparagine during germin-

ation has been reinvestigated. Capdevila & Dure (1977)

Figure 6 The accumulation of asparagine in spears of asparagus,

following harvesting. Data from Hurst et al. (1998), with permission.

DW, dry weight.

Figure 7 The accumulation of asparagine in the leaves of Arabidopsis

thaliana, placed in the dark for up to 6 days (1D–6D) or in the light

(C–C6D). Data from Lin & Wu (2004), with permission. FW, fresh weight.

Table 1 Nitrogen content of germinating Lupinus luteus seedlingsa

Ungerminated

Dry Seeds

6 Days

in Dark

15 Days

in Dark

15 Days in

Dark and

10 Days in Light

Protein N 8.72 5.49 1.71 1.78

Asparagine N 0 1.16 4.02 5.09

Basic N 0.46 0.97 1.22 1.03

Remaining N 0.16 1.72 2.39 1.40

Total N 9.34 9.34 9.34 9.34

aData expressed as N content per 100 g of ungerminated seeds, taken

from the original work of Schulze (1898), as reproduced by Chibnall

(1939).




5

showed that in germinating cotton (Gossypium hirsutum)

seedlings, the asparagine concentration increased from

280 nmol per cotyledon pair in the dry seed to 2885 nmol

after 5 days in the dark and 5362 nmol after 5 days in

the light. Slightly later, Elmore & King (1978), also

investigating germinating cotton, indicated that the major

site of asparagine accumulation was in the embryonic

axis, where during the 5 day germination period, the

soluble amino acid concentration increased from 4.7 to

723.7 lmol g21 DW. In mung bean (Vigna radiata), Kern

& Chrispeels (1978) demonstrated that the soluble amino

acid concentration increased rapidly during the first

5 days of germination and that asparagine accounted for

50% of the nitrogen in the cotyledon exudates, despite

the fact that the storage protein contained only 13.4% of

aspartyl residues.

In a very thorough experiment on loblolly pine (Pinus

taeda L.), King & Gifford (1997) initially incubated strati-

fied seeds at 2�C for 35 days, which was followed by

12 days of germination at 30�C. As the radicle emerged,

there was a pronounced change in the amino acid pro-

file of the seedling (Table 2). Despite the fact that the

storage protein of the megagametophyte contained high

concentrations of arginine (50% of total N), it can be

seen that after 12 days, the concentration of soluble

asparagine in the seedling greatly exceeded the other

major amino acids glutamine and arginine and ac-

counted for 70% of the total soluble pool. Rozan et al.

(2001) examined germinating seeds of a wide range of

lentil (Lens) species and showed that asparagine was

quantitatively by far the most important protein amino

acid in seedlings of all species studied. The concentration

of asparagine ranged from 18.96 to 62.24 mg g21 DW

following germination for 4 days, having increased dra-

matically from almost zero in the dry seed and repre-

sented 40–50% of the soluble amino acids. Even in

Canavalia ensiformis, a seed known to accumulate high

concentrations of the non-protein amino acid canava-

nine, asparagine was present as a higher percentage

of the total amino acid pool, 7 days after germination

(Camargos et al., 2004). However, it should be noted that

asparagine does not always accumulate during seed

germination (Lea & Joy, 1983), e.g. Cucurbita moschata (Chou

& Splittstoesser, 1972), Zea mays (Limami et al., 2002)

and coffee (Coffea arabica) (Shimizu & Mazzafera, 2000).

Stress and asparagine accumulation

Asparagine also accumulates under conditions of stress.

In some cases, this may be a direct biological response to

the stress conditions, for example, by contributing to the

maintenance of osmotic pressure. However, it may also be

an indirect result of the restriction of protein synthesis

under stress conditions.

Drought and salt stress

It is well established that the concentration of free proline

increases dramatically in plant tissues that have been

subject to drought or salt stress (Delauney & Verma, 1993;

Parry et al., 2005; Verdoy et al., 2006). However, there is

evidence that asparagine also accumulates to a consider-

able extent at the same time as proline (Stewart &

Larher, 1980), e.g. in soybean (Fukutoku & Yamada,

1984; King & Purcell, 2005), alfalfa (Fougere et al., 1991),

pearl millet (Kusaka et al., 2005) and wheat (Carillo

et al., 2005).

The amino acid contents of leaves of a salt-sensitive

wheat line were compared with a salt-tolerant amphidip-

loid, following treatment with increasing concentrations

of NaCl. Asparagine accumulated in the younger leaves

but not in the older, while proline exhibited the reverse

of this trend. In the young leaves, the concentration of

asparagine increased from 0 to 120 lmol g21 DW fol-

lowing treatment with up to 200 mM NaCl in a dose-

dependent manner (Fig. 8) while that of proline only

increased to 10 lmol g21 DW (Colmer et al., 1995). In

contrast, in barley the concentration of proline was five-

fold higher than that of asparagine, following treatment

with 450 mM NaCl, irrespective of the ages of the leaves

(Garthwaite et al., 2005). In the source photosynthetic

tissues of Coleus blumei, asparagine increased from 0.2 to

3.0 nmol g21 FW in 10 days following treatment with

60 mM NaCl and 12 mM CaCl2, a concentration second

only to that of arginine. However, in the sink non-

photosynthetic leaf tissue of C. blumei, the concentration

Table 2 Composition of the major soluble amino acids of loblolly pine

(Pinus taeda) seedlings during germination [data from King & Gifford

(1997) with permission]a

Amino Acid

Days

Dry 3 6 9 12

Aspartate 2.46 9.17 6.74 30.30 80.1

Glutamate 5.67 28.50 50.50 138 248

Serine 0.74 2.00 13.70 53.20 152

Asparagine 2.88 6.12 742 4388 12924

Glycine 0.25 0.75 1.15 4.7 ND

Glutamine 1.07 10.10 92.10 905 1699

Histidine 0.21 0.97 12.30 61.4 195

Threonine 0.22 0.78 5.82 17.3 45.5

Alanine 1.25 5.47 39.20 76.1 174

Arginine 4.29 15.20 141 755 2326

Proline 3.12 6.95 13.3 32.2 38.7

aDry seeds were initially imbibed at 2�C for 35 days and then incu-

bated at 30�C for the number of days shown. Data expressed as nmol

per seedling, taken from King & Gifford (1997). ND, not determined.




of asparagine increased from 1.0 to 9.0 nmol g21 FW

10 days after the addition of saline, which was twice that

of arginine (Gilbert et al., 1998).

Mineral deficiencies

With the exception ofmolybdenum,which is a constituent

of both nitrogenase and nitrate reductase (Schwartz &

Mendel, 2006), the growth of plants in a medium defi-

cient in an individual mineral ion, particularly if there is

a plentiful supply of nitrogen, can stimulate the accumu-

lation of high concentrations of asparagine. A review of

the early literature by Stewart & Larher (1980) indicated

that deficiencies in potassium, sulphur, phosphorus and

magnesium were well documented in a range of plants

and that for tomato, deficiencies of micronutrients, in

particular zinc, also stimulated large increases in aspar-

agine concentration (Possingham, 1956).

Although phosphate deficiency is frequently followed

by arginine accumulation (Rabe & Lovatt, 1986), again

there is evidence that asparagine also increases at the

same time. In soybean deprived of phosphorus for

20 days, considerable amounts of asparagine accumu-

lated in the roots and stems, while arginine accumulated

in the leaves (Rufty et al., 1993). Higher asparagine con-

centrations were detected in the root and in particular

the shoot of young tobacco plants deprived of a phospho-

rus supply for 10 days (Rufty et al., 1990). Similarly,

asparagine accumulated in the nodules and the roots of

white clover subjected to decreasing concentrations of

phosphorus (Almeida et al., 2000).

When plants are grown under sulphur-deficient condi-

tions, there is a reduction in the synthesis of the amino

acids cysteine and methionine and the antioxidant gluta-

thione (Nikiforova et al., 2006). In maize seeds, such

a deficiency in sulphur has been shown to give rise to an

alteration in the proportion of types of protein formed

and an increase in soluble asparagine to 50% of the total

pool (Baudet et al., 1986). In maize suspension culture

cells, the increase in asparagine accumulation following

sulphur starvation was shown to be as a result of de novo

synthesis and not protein hydrolysis (Amancio et al.,

1997). Similar increases in soluble asparagine in the

shoots of sulphur-deficient wheat varieties (Zhao et al.,

1996) and Italian rye grass (Mortensen et al., 1993) have

been observed.

Toxic metals

Plants exposed to toxic metals have been shown to accu-

mulate specific amino acids such as proline and histidine,

which may have a beneficial function (Sharma & Dietz,

2006). However, there is evidence that asparagine can

bind to cadmium, lead and zinc (Bottari & Festa, 1996).

It was suggested that in the grass Deschampsia cespitosa,

the asparagine which accumulated in the roots of ammo-

nium-grown plants formed an intracellular complex

with zinc and thereby decreased its toxicity (Smirnoff &

Stewart, 1987). White et al. (1981) were able to demon-

strate the presence of asparagine–copper complexes in

the xylem of soybean and to a lesser extent in tomato.

Cadmium is a major pollutant worldwide and is detri-

mental to plant growth (Benavides et al., 2005; Gratao

et al., 2005). For lettuce plants growing in axenic hydro-

ponic culture, cadmium caused an increase in asparagine

in both the roots and shoots (Costa & Morel, 1994).

Similar data of cadmium-induced asparagine accumula-

tion in roots and to a lesser extent in shoots were also

obtained in lupins (Fig. 9) (Costa & Spitz, 1997). Cadmium

induced an almost 10-fold increase in the asparagine

concentration of tomato roots but only fourfold in the

leaves. However, glutamine was still the major amino

Figure 8 The concentration of asparagine and proline (lmol g21 DW) in the youngest leaves of Triticum aestivum cv Chinese Spring (d) and Chinese

Spring x Lophopyrum elongatum amphidiploid (s) following treatment with increasing concentrations of NaCl for 18 days. Data from Colmer et al.

(1995), with permission. DW, dry weight.




7

acid being transported in the xylem and phloem and was

little affected by cadmium (Chaffei et al., 2004).

Pathogen attack

When detached tomato leaves were maintained under

sterile conditions for 10 days, the asparagine concentration

increased from 25.7 to 1150 nmol g21 FW. However, if

the leaves were infected with the bacteria Pseudomonas

syringea, the asparagine concentration rose to 19 843

nmol g21 FW, an 800-fold increase from when the

leaves were still on the plant (Perez-Garcia et al., 1998).

There was evidence that a novel form of cytosolic gluta-

mine synthetase was synthesised in the infected leaf

mesophyll cells, which was involved in the dramatic

increase in the rate of asparagine accumulation.

Following the infection of cocoa (Theobroma cacao) with

witches’ broom, the basidiomycete fungus Crinipellis perni-

ciosa, visible signs of chlorotic leaves and stem swelling were

observed after 21 days. Asparagine, which was already

present in high concentrations in the leaves, increased to

even higher levels after this time (Scarpari et al., 2005).

The enzymes of asparagine metabolism

Asparagine synthetase (AS)

The major route of asparagine synthesis involves the

initial assimilation of ammonia to the amide position of

glutamine (Lea & Miflin, 2003), followed by the transfer

to form the amide position of asparagine (Ta et al., 1986;

Rhodes et al., 1989). The enzyme AS (EC 6.3.5.4) cataly-

ses the adenosine triphosphate (ATP)-dependent transfer

of the amino group of glutamine to a molecule of aspar-

tate to generate glutamate and asparagine:

Glutamineþ Aspartateþ ATP/

Glutamateþ Asparagineþ AMPþ PPi

It has also been proposed that the enzyme can use

ammonia directly as a substrate (Oaks & Ross, 1984),

particularly if the concentration is high, but the in vivo

operation of this pathway has not been clearly demon-

strated. Aspartate is synthesised by transamination of

oxaloacetate and is also an important precursor of the

essential amino acids, lysine, threonine, methionine and

isoleucine (Azevedo et al., 1997, 2006; Ferreira et al.,

2006).

Asparagine may also be formed following the detoxifica-

tion of cyanide (Piotrowski & Volmer, 2006) and the

transamination of 2-oxosuccinamic acid (Joy, 1988), but

the fluxes through these pathways are not thought to be

of any significant magnitude (Sieciechowicz et al., 1988c).

In this article, we will concentrate on the enzyme AS.

Measuring the expected levels of activity of the enzyme

AS in plant tissues has proved a difficult task. On many

occasions, authors have reported very low or zero levels of

activity, although the particular plant tissue has been

shown to be synthesising asparagine at high rates (Kern

& Chrispeels, 1978; Joy et al., 1983; Brears et al., 1993;

Hurst & Clark 1993; Chevalier et al., 1996; Seebauer

et al., 2004). This inability to measure AS activity may

have been because of an inherent instability of the

protein, the presence of inhibitors or the competing

enzyme asparaginase. More recently, attempts have been

made to improve the extraction assay of AS in crude

plant extracts using a complex extraction buffer, con-

taining EDTA, MgCl2, ATP, aspartate, glycerol, DTT, and

b-mercaptoethanol, followed by detection of 14C-aspara-

gine using high performance liquid chromatography

(Romagni & Dayan, 2000).

Asparagine synthetase has been assayed and studied to

a limited extent in germinating seedlings (Streeter, 1973;

Lea & Fowden, 1975; Rognes, 1975; Dilworth & Dure,

1978), nitrogen-fixing root nodules (Scott et al., 1976;

Huber & Streeter, 1984, 1985; Shi et al., 1997) and maize

roots (Oaks & Ross, 1984) and leaves (Dembinski et al.,

1996). The enzymes isolated from these different sources

exhibited similar properties. Plant ASs have a broad pH

optimum from 7.5 to 8.3 and are inhibited by the gluta-

mine analogues azaserine and albizziine and by calcium

ions. The Km values vary between 0.8 and 3.6 mM for

aspartate, 0.04–1.0 mM for glutamine and 0.08–0.15 mM

Figure 9 The concentration of asparagine in the roots (s) and shoots

(d) of Lupinus albus following treatment with increasing concentrations

of cadmium for 15–17 days. Data from Costa & Spitz (1997), with

permission.




for Mg-ATP. There is also a requirement for chloride ions

for maximum activity (Rognes, 1980).

The purification to homogeneity of AS from alfalfa root

nodules has been reported (Shi et al., 1997), but no de-

tails of the methods used or the kinetic properties were

provided. Galvez-Valdivieso et al. (2005) were able to

express both genes encoding AS isolated from Phaseolus

vulgaris in Escherichia coli. The PVAS2-encoded protein

was used to raise antibody that recognised both the

P. vulgaris gene products with a molecular mass of

65 kDa. AS protein was detected in mature roots, sen-

escing leaves and only very early in the development of

the root nodules of P. vulgaris (Galvez-Valdivieso et al.,

2005). Again unfortunately, no attempt was made to

study the properties of the enzyme protein.

The genes encoding AS

The breakthrough in our understanding of plant AS came

with the isolation of two genes encoding the enzymes

AS1 and AS2 from peas by Tsai & Coruzzi (1990, 1991).

The two proteins of molecular mass 66.3 and 65.6 kDa

are highly homologous with the human enzyme and

have a purF glutamine-binding site at the NH2 terminus,

consisting of a Met-Cys-Gly-Ile sequence, indicating

that glutamine is the likely substrate in vivo (Richards &

Schuster, 1998). Northern blot analysis indicated that

expression of both genes is repressed by light in the

leaves but that in the roots, AS2 is expressed constitu-

tively and only AS1 is repressed by light. The repression

of the genes encoding AS by light and stimulation in the

dark agrees with the early work showing that asparagine

accumulation was stimulated by darkness.

An AS complementary DNA (cDNA) clone was isolated

from asparagus spears that encoded a 66.5 kDa protein

that was 81% identical to the AS1 from pea (Davies &

King, 1993). Expression of the gene was not detected in

the asparagus spears immediately on harvest, but after

6 h, there was a rapid induction of messenger RNA

(mRNA) synthesis along the spear, but expression was

not affected by light (Davies et al., 1996; Eason et al.,

1996). A similar induction of expression of AS was also

shown in harvested broccoli florets (Downs & Somerfield,

1997). It was proposed that the induction of AS mRNA

was stimulated by a rapid reduction in the soluble sugar

content. Although this proposal was confirmed using

callus cultures, it proved experimentally difficult in

excised asparagus spears (Irving et al., 2001). Later anal-

ysis of the promoter of the asparagus gene identified

a potential carbohydrate-responsive element at 2410 to

2401 bp relative to the translation initiation ATG, with

sequence identity to a rice a-amylase carbohydrate-

responsive element (Winichayakul et al., 2004a). Further

studies confirmed that low carbohydrate but not dark-

ness acted as the signal for the induction of the promoter

of asparagus AS (Winichayakul et al., 2004b), probably

through the involvement of hexokinase.

This simple story of carbohydrate regulation became

somewhat complicated when it was found that there

were three genes encoding AS in A. thaliana (Lam et al.,

1994, 1998), which appeared to be regulated in totally

different manners. Expression of ASN1 was stimulated

when plants were placed in the dark and dramatically

repressed following exposure to light for only 2 h, while

sucrose could to some extent substitute for light. In con-

trast, the expression of ASN2 was induced during the

same period and further stimulated for another 16 h in

the light; again sucrose could substitute for light. Even

more interestingly, the expression of ASN1 was stimu-

lated by the amino acids asparagine, glutamine and glu-

tamate, while ASN2 was repressed by the same amino

acids. Expression of the ASN3 gene was not detected in

any of the organs examined. Further studies in A.

thaliana by Thum et al. (2003) indicated that light was

able to override carbon in the regulation of ASN1, while

carbon was able to override light as the major regulator

of ASN2. There was also evidence that blue and red light

had differential effects on the expression of the AS

genes. Wong et al. (2004) went on to demonstrate in

A. thaliana that the light induction of ASN2 is ammo-

nium dependent. In addition to ammonium, stresses

such as salinity and cold also increased ASN2 mRNA lev-

els, and these stresses correlated with increases in

internal ammonium ions.

Three distinct genes encoding AS have also been iden-

tified in sunflower (H. annuus) (Herrera-Rodrıguez et al.,

2002, 2004, 2006). HAS1 and HAS1.1 were shown to be

light-repressed genes whose transcripts accumulated to

high levels in darkness. Light regulated the genes by

means of two different mechanisms, a direct one, via

phytochrome, and an indirect one, stimulating photo-

synthetic CO2 assimilation and the production of carbon

metabolites such as sucrose. The third AS gene of sun-

flower, HAS2, was regulated by light and carbon in an

opposite manner to that of HAS1 and HAS1.1. HAS2 was

more widely expressed and was stimulated by light and

by sucrose. HAS1 and HAS1.1 expression were depen-

dent on the presence of a nitrogen source, while HAS2

transcripts were still found in N-starved plants. High

ammonium levels induced all three AS genes and par-

tially reverted the sucrose repression of HAS1 and

HAS1.1 (Herrera-Rodrıguez et al., 2004).

To investigate the involvement of asparagine and AS

genes in the main nitrogen mobilisation processes in sun-

flower, the expression of HAS1, HAS1.1 and HAS2 genes,

as well as the synthesis of asparagine and other nitrogen




9

and carbon metabolites, were studied during germina-

tion and natural senescence of cotyledons and leaves

(Herrera-Rodrıguez et al., 2006). HAS2 was expressed

early in germination, and there was a correlation

between AS transcript level and asparagine accumula-

tion in the sunflower tissues. During leaf senescence, all

three genes were expressed, during which time there

was a reduction in sucrose content. Somewhat surpris-

ingly HAS1 and HAS1.1 were not expressed during coty-

ledon senescence.

As well as the four species described in some detail

above, genes encoding AS have also been investigated in

maize (Chevalier et al., 1996), Lotus japonicus (Waterhouse

et al., 1996), broccoli (Downs & Somerfield, 1997), soy-

bean (Hughes et al., 1997), alfalfa (Shi et al., 1997;

Carvalho et al., 2003), radish (Nozawa et al., 1999),

P. vulgaris (Osuna et al., 1999, 2001), Sandersonia aur-

antiaca (Eason et al., 2000), barley (Moller et al., 2003),

tomato (Olea et al., 2004), wheat (Wang et al., 2005) and

Pinus sylvestris (Canas et al., 2006). In most (but not all)

plants, a family of two genes has been identified.

Although there is considerable variation between plants

in the exact mechanisms involved in the regulation of the

expression of AS, there is an overall consensus. The

expression of one gene (often that which is most highly

expressed) is induced by a reduction in soluble carbohy-

drate supply and in some cases darkness, while a second

gene is more widely expressed but may be stimulated by

carbohydrate and light. An increased supply of reduced

nitrogen, either as ammonium or amino acids, induces

expression of AS genes.

Analysis of the amino acid sequences of plant ASs shows

that the proteins contain glutamine, aspartate and AMP

binding sites and are related to the E. coli asparagine syn-

thetase ASB glutamine-dependent enzymes (Canas et al.,

2006). Phylogenetic trees of the plant amino acid se-

quences have been constructed by a number of re-

searchers and compared with those of the bacteria, fungi

and animals (Shi et al., 1997; Osuna et al., 2001; Moller et

al., 2003; Canas et al., 2006). In the most recent study, the

plant sequences were clustered in two main groups: (a)

the sequences close to A. thaliana AS1 and (b) those grou-

ped with A. thaliana AS2 and AS3. The legume sequences

were located in the AS1 cluster, while the monocot

sequences were in the AS2/3 group (Canas et al., 2006).

Asparagine catabolism

There are two established pathways of asparagine catabo-

lism in higher plants, and these have been considered

in detail by Ireland & Joy (1981), Joy (1988) and

Sieciechowicz et al. (1988c). Asparagine can be trans-

aminated, particularly in leaves, to yield oxosuccinamic

acid, which may then be reduced to hydroxysuccinamic

acid and subsequently deamidated to yield malate. The

major form of asparagine, glyoxylate aminotransferase,

has been studied in detail in peas (Ireland & Joy,

1983a,b). It is likely that asparagine is metabolised

through the above route as part of the photorespiratory

nitrogen cycle (Murray et al., 1987; Keys, 2006) but that

the majority of the nitrogen is continuously recycled and

that there is little net catabolism of asparagine.

Asparaginase

Asparaginase (EC 3.5.1.1) catalyses the hydrolysis of

asparagine to yield aspartate and ammonia. The ammonia

is subsequently reassimilated by glutamine synthetase:

Asparagineþ H2O/Aspartateþ NH3

The assay of asparaginase has also proved difficult in

higher plants, with some plant sources providing extracts

with high rates of activity and others low or zero; a full

description of the early setbacks has been provided by

Sieciechowicz et al. (1988c).

It was the detailed investigation by Atkins et al. (1975)

that first gave an important indication of a reliable

source of plant material that would be useful for study-

ing asparagine metabolism. The investigators fed [14C]-

and [15N]-labelled asparagine to the shoots of L. albus

that were producing seeds inside pods. Most of the 15N

in the endosperm fluid was recovered as ammonia, glu-

tamine and alanine, while the 14C was not present in

amino acids. As the seed developed, both 15N and 14C

were found to be present in the amino acids of the seed

storage proteins. Particularly high asparaginase activity

was detected during the maturation development of the

L. albus cotyledons (Atkins et al., 1975). Subsequently,

a number of workers confirmed the presence of high

activities of asparaginase in legume seeds during the

maturation process (Lea et al., 1978; Murray & Kennedy,

1980; Chang & Farnden, 1981). An unusual twist to the

story came when Sodek et al. (1980) described the pres-

ence of an asparaginase in both the testa and maturing

cotyledons of peas that was totally dependent on the

presence of potassium ions. Gomes & Sodek (1984) then

went on to demonstrate that the asparaginase in devel-

oping soybean cotyledons was also K+ dependent. In

soybean cotyledons cultured in vitro, the level of activity

of the K+-dependent asparaginase was greatly reduced

by the supply of glutamine (Tonin & Sodek, 1990).

A K+-dependent asparaginase has also been studied in

some detail in pea leaves, where activity increases in the

light and decreases in the dark (Sieciechowicz et al.,

1985). The use of inhibitors of protein synthesis and

proteases demonstrated that this diurnal variation in




asparaginase activity was because of enzyme synthesis

in the light and proteolytic degradation in the dark

(Sieciechowicz et al., 1988a,b; Sieciechowicz & Ireland,

1989a). It was proposed that this type of regulation

ensured that the enzyme is only functional in the light

when there is sufficient ATP, and reducing power to fuel

the glutamate synthase cycle (Sieciechowicz et al.,

1988a,b).

The K+-independent asparaginases isolated from dif-

ferent lupin species were shown to have similar proper-

ties including Km values for asparagine (4–12 mM) and

pH optima (8.0–8.5) (Lea et al., 1978; Chang & Farnden,

1981). It was originally suggested that the native

enzyme from Lupinus polyphyllus seeds was a dimer of

molecular mass of 71–72 kDa with subunits of 35–38

kDa (Lea et al., 1978; Sodek & Lea, 1993). Lough et al.

(1992a) went on to show that when asparaginase was

purified from Lupinus arboreus seeds, although the native

molecular mass was 75 kDa, three polypeptides in the

range 14–19 kDa were present following sodium dodecyl

sulphate polyacrylamide gel electrophoresis (SDS-PAGE).

At the time, the reason for the multiple number of sub-

units was not clear. Sodek et al. (1980) reported a Km for

asparagine for the K+-dependent asparaginase in pea as

3.2 mM for the cotyledon and 3.7 mM for the testa

enzyme and a native molecular mass of 68 kDa. Similar

Km values were determined for the K+-dependent

enzyme in pea leaves but with a lower native molecular

mass of 58 kDa (Sieciechowicz & Ireland, 1989b).

The genes encoding asparaginase

A cDNA clone encoding a K+-dependent asparaginase

was isolated from L. arboreus. This encoded a 32.8 kDa

protein, which appeared to be only expressed at a spe-

cific time during seed maturation coinciding with high

enzyme activity. Somewhat surprisingly, the gene was

not expressed in roots, which had also been shown to

have high asparaginase activity (Chang & Farnden,

1981; Lough et al., 1992b). Dickson et al. (1992) isolated

a genomic sequence encoding asparaginase from Lupinus

angustifolius that contained four exons and three introns.

The 5#-flanking region contained sequences associated

with nodule-specific and seed-specific expression. A geno-

mic clone encoding asparaginase was also isolated from

A. thaliana, encoding a protein with a predicted mole-

cular mass of 33 kDa (Casado et al., 1995).

The promoter of the asparaginase gene isolated by

Dickson et al. (1992) was ligated to a b-glucuronidase(GUS) reporter gene and transformed into tobacco plants

(Grant & Bevan, 1994). GUS activity was found mainly

in the developing tissues of mature plants such as apical

meristems, expanding leaves, inflorescences and seeds of

tobacco. The chimaeric gene was also used to investigate

transient expression in lupins. As might be expected

from earlier enzyme measurements, transient GUS ex-

pression was detected in the developing pods, seed testas

and cotyledons.

Our understanding of the molecular structure of plant

asparaginases took a leap forward when Hejazi et al.

(2002) were able to express the A. thaliana gene in

E. coli. The purified asparaginase protein was shown to

comprise peptides of approximately 35, 24 and 12 kDa,

following SDS-PAGE. The authors proposed that the two

smaller peptides were the result of proteolytic cleavage

and that the native protein rather than being a dimer

was in fact an (ab)2 tetramer. Analysis of the substrate

specificity of the recombinant A. thaliana protein showed

that the enzyme could use a range of b-aspartyl peptidesas substrates, with b-aspartyl-phenylalanine and b-aspartyl-alanine having Vmax values close to that of asparagine.

Borek et al. (2004) expressed a gene encoding the L.

luteus K+-independent asparaginase (Borek et al., 1999)

in E. coli. The recombinant native enzyme had a molecu-

lar mass of 75 kDa, but the translated peptide under-

went an autoproteolytic cleavage leading to the

formation of two subunits, 23 kDa (a subunit) and

14 kDa (b subunit), confirming the existence of the

(ab)2 tetramer. This cleavage gives rise to a N-terminal

nucleophilic threonine residue on the b subunit. Phylo-

genetic analysis of N-terminal nucleophilic hydrolases

indicated that the amino acid sequences of the plant as-

paraginases from A. thaliana, L. luteus, barley, rice and

soybean fell in a group with bacterial enzymes that also

had isoaspartyl peptidase activity. Although asparagine

was a substrate for the recombinant L. luteus enzyme

with a Km of 4.8 mM, the surprising result was that b-as-partyl-leucine was a substrate with more than four times

the Vmax and a Km of only 0.14 mM (Borek et al., 2004).

Michalska et al. (2006) crystallised the K+-independent

asparaginase from L. luteus and carried out a detailed

analysis of the quaternary structure. The protein ex-

hibited a abba fold typical of N-terminal nucleophilic

hydrolases. Each of the two active sites of the (ab)2heterotetrameric protein is located in a deep cleft

between the b-sheets, near the nucleophilic threonine

193 residue, which is liberated in the autocatalytic event

at the N-terminus of the b subunit. A comparison of the

active sites of the L. luteus asparaginase and the E. coli

EcAIII enzyme showed a high degree of conservation of

the residues participating in substrate/product binding

and of all other residues forming important hydrogen

bonds within the catalytic pocket. Some evidence was

provided as to how the active site could accept both

asparagine and b-aspartyl peptides.




11

The availability of the complete sequence of the

A. thaliana genome allowed Bruneau et al. (2006) to

isolate a second gene encoding an asparaginase enzyme

that was dependent on K+ for full activity. The K+-

dependent enzyme had 55% identity with the K+-

independent form, indicating that they belong to two

evolutionarily distinct subfamilies of plant asparaginases,

as revealed by phylogenetic analysis. However, the two

enzyme proteins had remarkably similar structures, the

K+-dependent enzyme having a subunits of 22.7 kDa

and b subunits of 13.6 kDa. In addition, there were

conserved autoproteolytic pentapeptide cleavage sites

commencing with the catalytic threonine nucleophile, as

determined by electrospray ionisation-mass spectrome-

try (ESI–MS) analysis. The K+-dependent enzyme in A.

thaliana had a lower Km and much higher Vmax than the

K+-independent form, indicating an 80-fold higher cata-

lytic efficiency with asparagine. The K+-dependent enzyme

was unable to use b-aspartyl dipeptides as substrates, dem-

onstrating a clear difference between the enzyme and the

K+-independent enzyme (Bruneau et al., 2006).

The steady-state mRNA levels of the two asparaginase

genes in A. thaliana were determined by quantitative

reverse transcriptase polymerase chain reaction (RT-PCR)

in various tissues during development. As expected, the

expression of both genes was associated with sink tissues

and was highest in flowers, siliques, flower buds and

leaves. The two genes showed largely overlapping pat-

terns of developmental expression, but in all the tissues

examined, the transcript levels of the K+-dependent

enzyme were lower than those of the K+-independent

enzyme. Microarray analysis showed that the K+-depen-

dent enzyme was highly expressed in stamens and

mature pollen of A. thaliana (Schmid et al., 2005).

Bruneau et al. (2006) suggested that as the spatial pat-

terns of the expression of the two genes were largely

overlapping, the two enzymes had redundant functions.

As mutants and knockout lines are not currently avail-

able, it is not possible to test this hypothesis. However,

the key question is why should plants have one form of

an asparaginase which apparently has a greater activity

and a higher affinity for isoaspartyl peptides? One pos-

sible reason is because of the frequently occurring con-

version of asparagine to isoaspartyl residues in mature

proteins. This is a dangerous modification, as it causes a

structural change that may significantly alter the three-

dimensional structure of the protein, leading to a change

of activity, degradation or aggregation. Proteins with

isoaspartyl residues can be degraded by proteolytic en-

zymes, but among the products, there will be b-aspartylpeptides containing N-terminal isoaspartyl residues which

require specialised hydrolytic enzymes (Clarke, 2003;

Shimizu et al., 2005). Borek et al. (2004) proposed that

isoaspartyl peptidase activity could be particularly impor-

tant in seeds that have to retain their ability to grow for

a very long time. During the storage period, the seed

proteins can undergo modification, and isoaspartyl pep-

tidase activity is necessary to destroy the altered proteins

and to allow only the healthy seeds to germinate.

Asparagine in crop plants

The accumulation of asparagine in the edible organs of

crop plants is of particular interest in relation to acrylam-

ide formation in food (discussed below). Current informa-

tion indicates that both genetic and environmental factors

may be important, with the latter being of particular con-

cern in relation to the sourcing of low asparagine cereals

and potatoes for food processing.

Cereal seeds

There are relatively few detailed studies of free amino

acids in cereal seeds, with most emphasis being on the

protein fractions which determine nutritional quality

and functional properties. Further, many early studies

often determined free amino acids as part of the ’non-

protein nitrogen’ (NPN) fraction that would include

other water-soluble, low molecular weight nitrogenous

components including peptides. Thus, Byers et al. (1983)

showed that the NPN extracted with three concen-

trations of NaCl solution accounted for 6.4–6.7% of the

total grain N of wheat, and similar values of 5–6% were

reported for three wild type (i.e. non-mutant) barley

lines by Køie & Doll (1979). In maize, total free amino

acids have been determined as 4.4% and 2.9% of the

total grain nitrogen in two hybrids with normal grain

texture (Sodek & Wilson, 1971). Even less data are avail-

able on the contents of asparagine in these fractions, but

values of 3.31% of the total free amino acids have been

reported for five inbred rye lines with low protein con-

tent (Dembinski & Bany, 1991) and 7.9% for a single

inbred line of maize (Sodek & Wilson, 1971). Hence, it

can be concluded that free amino acids generally

account for about 5% or less of the total nitrogen in

cereal grains, and asparagine accounts for a low pro-

portion (certainly less than 10%) of this fraction. How-

ever, these values may be dramatically affected by both

genetic and environmental factors.

High-lysine mutants

A number of high-lysine mutants have been identified in

cereals, notably maize, barley and sorghum (see reviews

by Bright & Shewry, 1983; Shewry et al., 1987; Coleman &

Larkins, 1999; Azevedo et al., 2004; Ferreira et al., 2005).




In most cases, the high-lysine phenotype results, at

least in part, from a decrease in the lysine-poor storage

protein (prolamin) fraction and compensatory increases

in other more lysine-rich fractions including free amino

acids.

Køie & Doll (1979) analysed 11 induced high-lysine

mutants in the barley cultivars Bomi and Carlsberg II

and showed that these had up to threefold increases in

free amino acids (expressed as mg g21) and up to two-

fold increases in NPN. However, amino acid analyses

of these fractions were not reported. More extensive

studies have been reported of maize mutants, showing

substantial increases in free amino acids but that the

magnitude of these varies between mutant lines and re-

ports (Murphy & Dalby, 1971; Ma & Nelson, 1975; Misra

et al., 1975; Arruda et al., 1978). One of the most detailed

studies was reported by Sodek & Wilson (1971). They

showed that free amino acids were increased from 4.4%

to 19.7% of the total N in an opaque2 line in the R802

background, with other normal (WF9�M14) and opa-

que2 (R802�R75 opaque2) lines having 2.9% and 9.1%

free amino acids, respectively. In contrast, the amount of

free amino acids was lower in the R802 floury2 line

(2.2%) than in the normal R802 background (4.4%).

These authors also determined the amino acid compo-

sitions of free amino acid fractions from the R802 and

R802 opaque2 lines, showing 7.9% and 7.2% asparagine,

respectively. Similarly, Arruda et al. (1978) compared

developing endosperms of a double mutant (sugary1/

opaque2) line of maize with the normal background line.

Total free amino acids and asparagine were determined

as lmol per endosperm, and the ratios of these in the

mutant : normal endosperms were similar, 1.7:1 for free

amino acids and 1.85:1 for asparagine. Thus, it appears

that there is little or no impact of the mutations on the

proportion of asparagine in the fractions.

Impact of nutrition

Winkler & Schon (1980) grew barley plants in pots

under glasshouse conditions with four nutrient regimes:

0.6 g N per pot, 1.2 g N per pot as single and split appli-

cations and 1.8 g N per pot as a split application

(Table 3). Although the total free amino acids and total

asparagine increased with grain nitrogen, the proportion

of asparagine remained constant at about 15% of the

fraction. Thus, increasing seed N did not disproportion-

ally affect asparagine accumulation.

However, subsequent work on barley by Shewry et al.

(1983) indicated that the ratio of S:N was an important

determinant of asparagine accumulation rather than N

alone. They grew plants under sulphur-deficient con-

ditions, which restricted their ability to synthesise the

major hordein (prolamin) storage proteins that contain

cysteine and methionine. Under these conditions, total

hordein was decreased from 51.0% to 27.0% and from

46.7% to 26.9% of the total seed N in two cultivars, and

NPN increased from 6.8% to 29.1% and from 7.3% to

32.6%, respectively. The amino acid composition of the

NPN fraction was not determined, but analysis of total

grain demonstrated increases in the proportion of aspar-

tate + asparagine to 19.2% and 18.5% of the total

amino acids from 5.7% and 5.3%, respectively. The

authors suggested that this resulted from the accumula-

tion of free asparagine that acted as an alternative nitro-

gen store when the ability to synthesise hordein was

compromised.

When the study of Shewry et al. (1983) was carried

out, the results were of little more than academic inter-

est as sulphur deficiency was not an issue for cereal cul-

tivation, except in some specific parts of the world

outside Europe. This situation has now changed, with

increasing areas of land in Western Europe becoming

sulphur deficient as a result of decreased use of sulphur-

containing fertilisers and reduced atmospheric deposi-

tion (Zhao et al., 1999). In fact, the Home Grown Cereals

Authority currently estimates that 23% of the UK is at

risk of sulphur deficiency for cereal cultivation (www.

hgca.uk; verified 3/11/2006). Consequently, Muttucumaru

et al. (2006) have carried out a detailed study of wheat

grown in glasshouse trials and in a field trial on the sul-

phur-deficient site at Woburn (Bedfordshire, UK). This

showed that similar high levels of asparagine accumula-

tion (75 mmol kg21 FW) occurred in grain grown in the

field as in the highly sulphur-deficient grain grown in

glasshouse experiments (48–153 mmol kg21 FW) (Table 4).

The implications of these levels for the formation of

acrylamide during processing are discussed below.

Other environmental factors

Baker et al. (2006) reported detailed metabolomic com-

parisons of a series of transgenic and control lines of

wheat grown on two sites (Long Ashton, near Bristol,

Table 3 Total grain N, free amino acids and free asparagine in barley

grain grown with various levels of nitrogen [data from Winkler &

Schon (1980), with permission]

Fertilisation (g N per pot) 0.6 0.6 + 0.6 1.2 1.2 + 0.6

Total grain N (%) 1.58 2.18 1.90 2.54

Total free amino acids

(lmol per 100 mg flour)

1.066 1.747 1.121 2.057

Free asparagine

(lmol per 100 mg flour)

0.150 0.278 0.146 0.300

Free asparagine

(% total free amino acids)

14.1 15.9 13.0 14.6




13

and Rothamsted, near London, UK) over 3 years. These

included the analysis of free amino acids in samples

grown in 2000 using gas chromatography-mass spec-

trometry (GC–MS) analysis. The transgenic lines had

been engineered to express additional copies of gluten

protein genes, and there were few differences between

the amounts and compositions of free amino acids pres-

ent in the control and transgenic lines. However, there

were consistent differences between sites, with the levels

of asparagine (Fig. 10) and several other free amino

acids (aspartic acid, c-aminobutyric acid, glutamine and

glutamic acid) (results not shown) all being higher in the

grain grown at Rothamsted than in that grown at Long

Ashton.

These differences could not be accounted for based on

the N and S contents of the grain, with the samples from

Long Ashton having mean contents of 1498 mg S kg21

and 2.45% N on a DW basis and those from Rothamsted

1710 mg S kg21 and 2.73% N (unpublished results from

the study). The N:S ratios for the two sites were there-

fore 16.36 for Long Ashton and 15.97 for Rothamsted,

with Long Ashton being slightly more sulphur deficient.

The two sites also differed in soil type and climatic con-

ditions (temperature, precipitation), but the reason for

the effect on asparagine accumulation has not been

established.

Grain protein content

Dembinski & Bany (1991) determined the free amino

acids in five inbred lines of rye with normal protein con-

tent (’13% DW) and five lines with high protein con-

tent (’20% DW). The high-protein lines contained

about three times more free amino acids than the

normal-protein lines (124.1 lmol g21 DW compared with

45.4 lmol g21 DW), while asparagine increased from

3.31% to 17.37% of the pool of free amino acids. The

plants were grown with a modest level of nitrogen fertil-

iser (40 kg ha21), but sulphur was not added. Hence, the

increased accumulation in the high-protein lines could

possibly be accounted for by the increased demand for

nitrogen, resulting in sulphur deficiency.

Location within the grain

Fredriksson et al. (2004) determined the content of free

asparagine in a range of milling fractions from grain of

wheat and rye. The wholemeal flour from these species

contained 0.51 and 0.48 g asparagine kg21 DW for two

batches of wheat and 1.07 g kg21 DW for one sample of

rye. In both cases, the sieved flour contained less aspara-

gine (0.14, 0.17 for wheat; 0.53, 0.68 for rye) and the

bran contained more (1.48 for wheat; 2.61, 3.18 for rye).

However, the highest levels were present in wheat germ,

Table 4 Free asparagine (mmol kg21 FW) in grain samples from

wheat grown in pots under glass with adequate and deficient levels of

sulphur or in the field at the Rothamsted farm site at Woburn, Bed-

fordshire, UK, with the addition of sulphate fertiliser to give 0, 10 or

40 kg sulphur per hectare as indicated; the soil at this site has very

poor nutrient retention and without the addition of fertiliser is

severely sulphate deficienta

Cultivar Solstice Malacca Clare Hereward

Glasshouse + S 4.54 (0.50) 5.20 (1.10) 4.12 (0.26)

Glasshouse 2 S 75.4 (3.80) 153 (1.25) 47.9 (1.14)

Field 0 kg S 75.7 (2.62)

Field 0 kg S 55.7 (1.77)

Field 10 kg S 7.80 (0.29)

Field 40 kg S 4.43 (0.11)

Field 40 kg S 3.07 (0.23)

FW, fresh weight.aEach row in the table of field data represents a different plot. Data

from Muttucumaru et al. (2006).

Figure 10 The concentrations of asparagine (lg g21 fresh weight) in white flours of 48 transgenic and non-transgenic wheat lines grown in the field

at Long Ashton Research Station (Bristol, UK) (lanes 1–24 on the left) and Rothamsted Research (Harpenden, Herts, UK) (tracks 25–48 on the right).

Data from Baker et al. (2006), with permission.




with two fractions containing 4.88 and 4.99 g aspara-

gine kg21 DW.

Potato tubers

A number of recent studies have followed the amino acid

and sugar content of potatoes during storage. Generally,

storage at 2–20�C for severalmonths had little effect on the

free asparagine content, and where different cultivars

were compared, all responded in a similar way (Chuda

et al., 2003; Olsson et al., 2004; De Wilde et al., 2005;

Ohara-Takada et al., 2005; Matsuura-Endo et al., 2006)

(Table 5). However, after prolonged storage, protein

degradation can lead to an increase in both the amount

of free asparagine and its contribution to the total free

amino acid pool (Brierley et al., 1997). The relative sta-

bility of amino acid content during storage at low tem-

peratures contrasts markedly with large increases seen

in glucose, fructose and sucrose concentrations (Olsson

et al., 2004; De Wilde et al., 2005; Ohara-Takada et al.,

2005; Matsuura-Endo et al., 2006).

In potatoes, asparagine has been reported to be the

dominant free amino acid (33–59% as a percentage of

the total free amino acids) (Eppendorfer & Bille, 1996).

However, there are relatively few systematic studies that

compare the effects of genotype or environment on free

amino acids in tubers. In those experiments that have

been conducted and that compared tubers from different

cultivars grown at the same site, the asparagine contents

vary within a comparatively narrow range, e.g. 1.54–

1.93 mg g21 FW in commercially important Belgian cul-

tivars (De Wilde et al., 2005); 0.9–2.0 mg g21 FW in two

crisp processing and one table cultivar (Matsuura-Endo

et al., 2006); 3.8–5.3 mg g21 DW in three crisp processing

cultivars (Olsson et al., 2004). This contrasts dramatically

with those studies that compare tubers purchased from

local markets. Vivanti et al. (2006) reported almost 50-

fold variation (1.2–57.6 mg g21 FW) in the asparagine

content of tubers for 31 (9 Italian and 22 American) cul-

tivars purchased at markets. The extent to which this

variation in asparagine content is because of genotype

or other environmental factors is unclear, although the

data presented above suggest that it is unlikely to be

because of storage conditions. Unfortunately, there are

relatively few detailed studies on the effects of nutrition

or other environmental factors on the asparagine con-

tent of tubers. Several authors have reported that tubers

have increasing asparagine concentrations in response

to raised nitrogen fertilisation (Hoff et al., 1977; Amrein

et al., 2003; Silva & Simon, 2005). In a more recent study,

De Wilde et al. (2006) analysed tubers from three differ-

ent cultivars of potatoes grown with three nitrogen re-

gimes. A re-analysis of their data (Fig. 11) demonstrates

that both the free asparagine content and the total free

amino acid content were strongly positively correlated

with the N availability (r2 of 0.88 and 0.75, respectively),

although the percentage contribution of asparagine to

the total amino acid pool remained almost constant. Ear-

lier work by Osaki et al. (1995) showed that the form of

nitrogen supply, ammonia or nitrate did not affect the

free asparagine content of tubers. There are few system-

atic studies describing how the free amino acid content

responds to changes in other major nutrients (P, K and

S) or to environmental factors; a rigorous systematic

analysis of these responses is needed.

Table 5 Examples of the asparagine content of potato tubers after storage at 2–20�C for 2–52 weeks

Cultivar Temperature (�C) Time (Weeks) Amount

% Total

Amino Acid Pool Author

Toyoshira 2 2 0.7 mg g21 FW – Chuda et al. (2003)


Toyoshira 2 2 1.2 mg g21 FW – Ohara-Takada et al. (2005)

4 1.1 mg g21 FW

Bintje 4 24 16 mg g21 DW 35.4 De Wilde et al. (2005)

Pentland Dell 5 25 7.3 mg g21 FW 39.7 Brierley et al. (1997)

Bintje 8 24 17 mg g21 DW 37.7 De Wilde et al. (2005)

Pentland Dell 10 25 9.3 mg g21 FW 49.6 Brierley et al. (1997)

Snowden 10 2 0.9 mg g21 FW – Matsuura-Endo et al. (2006)

4 1.1 mg g21 FW –

18 1.0 mg g21 FW –

Toyoshira 18 2 1.1 mg g21 FW – Ohara-Takada et al. (2005)

4 1.4 mg g21 FW

Snowden 18 2 1.2 mg g21 FW – Matsuura-Endo et al. (2006)

4 1.6 mg g21 FW –

18 1.1 mg g21 FW –


DW, dry weight; FW, fresh weight.




15

The relationship between asparagine content and

acrylamide formation during processing

It only recently became apparent that the accumulation of

asparagine in the harvested organs of crop plants had im-

plications for food safety. A study published in 2002 by

a research group led by Margareta Tornqvist at the

University of Stockholm found that people had a signifi-

cant intake of acrylamide from cooked foods (Tareke et al.,

2002). Later that year, research at the University of

Reading provided the first evidence that acrylamide can

be generated from food components during heat treat-

ment as a result of the Maillard reaction (Fig. 12), which

occurs between amino acids and reducing sugars and

that asparagine was the amino acid required for its for-

mation (Table 6) (Mottram et al., 2002). Other work has

confirmed these findings (Stadler et al., 2002; Becalski

et al., 2003; Zyzak et al., 2003). The International Agency

for Research on Cancer has classified acrylamide as

’probably carcinogenic to humans’ (IARC, 1994). Carci-

nogenicity to humans has not been demonstrated in

epidemiological studies (Mucci et al., 2003), but car-

cinogenic action in rodents has been demonstrated. At

high doses, acrylamide also has neurological and repro-

ductive effects. Those foods with the highest levels of

acrylamide are carbohydrate-rich foods that have been

cooked at high temperatures, such as those achieved

during frying or baking (Table 7). They include foods

derived from wheat, maize, rye, other cereals and

potato. Levels of acrylamide can exceed 1000 parts per

billion (ppb); for comparison, the tolerance level for

water set by the World Health Organization is 1 ppb. To

date, no tolerance levels have been established for food,

and, with the exception of Germany, where the authori-

ties work with a de facto limit of 1000 ppb, no country

has yet specified maximum levels of acrylamide permit-

ted in food products. However, the European Commis-

sion Scientific Committee on Food has recommended

that levels of acrylamide in food should be as low as can

be reasonably achieved. Note that acrylamide is not

found in boiled or unheated foods and is only formed in

low concentrations in heated protein-rich foods, such

as meat.

N available kg ha-1

Amin

o ac

id c

onte

nt%

DW

250250150100500

0

1

2

3

4

5

Figure 11 The free asparagine (d) and total amino acids (s) in potato

tubers grown with different nitrogen availability [replotted from De

Wilde et al. (2006)]. DW, dry weight.

Figure 12 Acrylamide formation from asparagine and glucose via the

Maillard reaction [taken from Friedman (2003), with permission].

Table 6 Acrylamide produced in reactions at 175�C between glucose

and amino acids [based on data in Mottram et al. (2002), with

permission]

Amino Acid

Acrylamide (mg mol21 amino acid)

With Water Dry

Asparagine 221 25

Methionine Not detected 5

Glycine Not detected Not detected

Cysteine Not detected Not detected

Glutamine Trace Trace

Aspartic acid Not detected Not detected




The detection of acrylamide in foods led to much effort

worldwide by the food industry and regulatory authorities

to monitor acrylamide levels and to find means of reduc-

ing the levels and thereby any possible health risks. The

formation of acrylamide has been shown to be dependent

on time and temperature of cooking (Amrein et al., 2003;

Jung et al., 2003; Rydberg et al., 2003; Surdyk et al.,

2004; Taubert et al., 2004), and progress has been made

in some sectors of the food industry to reduce acrylam-

ide formation by changing processing methods. Potato

snack manufacturers, for example, claim to have

reduced acrylamide levels in their products by approxi-

mately 40%, although the evidence for this is anecdotal.

They also benefited from the fact that potato varieties

bred for crisp and chip production had been selected for

low sugar content for many years before the acrylamide

issue arose to ensure the correct colour after cooking.

However, the Maillard reaction is also responsible for the

characteristic colour and flavour of roast, baked and

fried foods, and any general inhibition of the Maillard

reaction to lower acrylamide levels is likely to affect col-

our and flavour quality. If sugars levels are too low, such

as may be achieved in potato products by washing

before cooking, then flavour and colour are affected.

The demand for fried, roasted and baked foods is

unlikely to diminish, and improvements brought about

by changes in processingmethods will have a limit. Atten-

tion must turn to the raw material, and several studies

have shown that the levels of precursors in the raw

material are an important factor (Amrein et al., 2003;

Biedermann-Brem et al., 2003; Grob et al., 2003; Haase et

al., 2004). Acrylamide in food is therefore a plant and

agricultural science as well as a food science issue, and

plant scientists, breeders and farmers must be engaged in

addressing it. Muttucumaru et al. (2006) demonstrated

a link between one agronomic parameter, sulphate

availability, and asparagine accumulation in wheat

grain, while Fig. 10 shows clear effects of location (Baker

et al., 2006). These results suggest strongly that plant-

and agronomy-based studies could make a significant

contribution in reducing the levels of acrylamide in pro-

cessed foods by improving the raw material.

The data reported by Muttucumaru et al. (2006) can be

used to show a clear relationship between wheat grain

asparagine and acrylamide formation during processing

(Fig. 13). Strategies for lowering acrylamide levels in

wheat and almost certainly other cereal products there-

fore need to reduce asparagine while keeping sugar at

a level sufficient to maintain flavour and colour quality.

The link between asparagine levels and acrylamide for-

mation in potato is not so clear; reports by Ohara-Takada

et al. (2005) and Wicklund et al. (2006), for example,

suggested that sugars not asparagine levels were limiting

in potato. However, in our view, more data are required

before definite conclusions can be drawn because varie-

ties may differ; it would be inappropriate at this point to

recommend to breeders that they should focus their ef-

forts entirely on sugars. We note that crisp manu-

facturers already use very low sugar varieties and still

have acrylamide levels in their products of several hun-

dred ppb. In varieties such as these, asparagine content

may be a more useful target, but there are insufficient

data available at this time to be certain.

Conclusions

Asparagine clearly plays a central role in nitrogen storage

and transport in plants, which is facilitated by its chemical

properties. This includes accumulation in a range of tissues

(sometimes transiently) and under stress conditions,

including conditions where the plant is unable to support

a normal level of protein synthesis. The lattermay account

in part for the wide range in asparagine concentrations in

harvested cereals and potatoes, and the consequences of

this account for the formation of acrylamide in foods.

However, there are clearly also genotypic differences,

Table 7 Acrylamide concentrations reported in some cooked foods

and food products [data from Friedman (2003)]

Food Product Acrylamide [lg kg21 (ppb)]

French-fried potato 200–12000

Potato chips, crisps 170–3700

Biscuits, crackers 30–3200

Snacks, other than potato 30–1915

Gingerbread 90–1660

Cereals, breakfast 30–1346

Crispbread 800–1200

806040200

0

1000

2000

3000

4000

5000

6000

Asn mmol kg-1 FW

Acry

lam

ide

µg. k

g-1

Figure 13 Correlation between asparagine in flour and acrylamide on

baking; samples taken from field-grown wheat; r2 = 0.998 [replotted

from Muttucumaru et al. (2006), with permission]. FW, fresh weight.




17

and to unravel these and how they interact with environ-

mental factors is an important topic for future research.

Efficient and reliable transformation systems are

now available for all major crops (Howarth et al., 2005;

Shewry & Jones, 2005; Meiyalaghan et al., 2006) includ-

ing cereals and potatoes, which are of greatest concern to

food processors in relation to acrylamide formation.

Determination of the factors and mechanisms that de-

termine asparagine accumulation may therefore ulti-

mately be manipulated to give safer food products for

consumers.

Acknowledgements

Rothamsted Research receives grant-aided support from

the Biotechnology and Biological Sciences Research

Council of the UK. We are indebted to the Conselho

Nacional de Desenvolvimento Cientıfico e Tecnologico

(CNPq-Brazil) for funding collaboration between L. S.

and P. J. L.

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Asparagine in Plants