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The content and quality of protein in winter wheatgrains depending on sulphur fertilizationMalle Järvan a , Liina Edesi a & Ando Adamson aa Department of Plant Sciences , Estonian Research Institute of Agriculture , Saku ,EstoniaAccepted author version posted online: 13 Apr 2012.Published online: 14 May 2012.

To cite this article: Malle Järvan , Liina Edesi & Ando Adamson (2012) The content and quality of protein in winter wheatgrains depending on sulphur fertilization, Acta Agriculturae Scandinavica, Section B — Soil & Plant Science, 62:7, 627-636,DOI: 10.1080/09064710.2012.683495

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ORIGINAL ARTICLE

The content and quality of protein in winter wheat grains depending onsulphur fertilization

MALLE JARVAN, LIINA EDESI & ANDO ADAMSON

Department of Plant Sciences, Estonian Research Institute of Agriculture, Saku, Estonia

AbstractOn soils lacking in water-soluble sulphur, the sulphur fertilization of winter wheat, in general, increases the yields. There arenot sufficient investigations about the influence of sulphur on the quality of yield. The objective of this work was toinvestigate the content and quality of protein in wheat grain depending on sulphur fertilization. The present study relies onfield trials conducted on two different soils during 2004�2009. Sulphur was applied with NS-fertilizer Axan or Axan Superat the rate of S 10 or 13.6 kg ha�1 accompanied by a nitrogen background of N 100 kg ha�1. The rates of N- and NS-fertilizers were divided and applied at the beginning and at the end of tillering. At harvest, the grain samples from trialvariants in four replications were taken, and the contents of crude protein, wet gluten, amino acids (lysine, threonine,cysteine, methionine) and gluten index in wheat grain were determined. Besides, the contents of amino acids wererecalculated on their concentrations in protein. The protein and wet gluten contents in grain varied significantly dependingon weather conditions of the trial years. On break-stony soil, sulphur increased the yield by 1.16 t ha�1 on average, i.e. by21.7%. With increasing yields the protein and wet gluten concentrations in grain decreased. Under the influence of sulphur,the gluten index increased significantly � from 58 to 74, i.e. by 27.6%. In 2004 and 2005, sulphur increased the cysteine andmethionine content in wheat grain. Although sulphur application in many cases decreased the protein and wet glutencontents in wheat grain, it improved the biological quality of protein because the concentrations of above-mentioned aminoacids recalculated on their concentrations in protein increased significantly. The sulphur application in pseudopodzolic soilhad a weaker effect on the grain quality than in break-stony soil.

Keywords: Crude protein, cysteine, gluten index, grain yield, lysine, methionine, threonine, Triticum aestivum, wet gluten.

Introduction

Sulphur was rarely deficient for agricultural crops

until about two decades ago, and has become one of

the most limiting nutrients for agricultural produc-

tion in Europe today (McGrath 2003, Loudet 2008,

Reinbold et al. 2008). Since the 1980s, sulphur

fertilization and the effect of sulphur deficiency on

growing plants, crops, and the properties of dough

prepared from wheat flour with sulphur deficiency

have attracted considerable interest (Reinbold et al.

2008).

Many investigations have shown a great signifi-

cance of sulphur for wheat production because the

application of sulphur fertilizers has influenced both

the yield and quality of wheat grain (Randall et al.

1990, Zhao et al. 1999, McGrath 2003, Jarvan et al.

2006, 2009, Weber et al. 2008, Mars 2009).

Several studies (Byers and Bolton 1979, Podlesna

and Cacak-Pietrzak 2008, Habtegebrial and Singh

2009, Mars 2009) indicated that the application

of sulphur-containing fertilizers increased both the

yield and the protein content of grain. However, in

some studies (Jarvan et al. 2006, Weber et al. 2008)

higher grain yields were correlated with lower protein

content. Depending on soil and weather conditions,

as well as the influence of applied sulphur fertilizers

the correlation between the grain yield and protein

content may be positive or negative (Jarvan et al.

2009). The effect of applied sulphur on the wheat

yield and grain quality is closely related to nitrogen

supply (Fitzgerald et al. 1999, Flaete et al. 2005,

Gyori 2005, Thomason et al. 2007, Jarvan 2008).

One of the major components of the storage

proteins in wheat grain are prolamins, accounting

for about 50% of the total grain N (Shewry et al.

Correspondence: M. Jarvan, Estonian Research Institute of Agriculture, Teaduse St. 13, EE75501 Saku, Estonia. E-mail: malle.jarvan@eria.ee

Acta Agriculturae Scandinavica Section B � Soil and Plant Science, 2012; 62: 627�636

(Received 1 February 2012; revised 2 April 2012; accepted 3 April 2012)

ISSN 0906-4710 print/ISSN 1651-1913 online # 2012 Taylor & Francis

http://dx.doi.org/10.1080/09064710.2012.683495

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1997). Prolamins are further divided into gliadins

and glutenins. Gliadins are minomers that are

soluble in aqueous alcohol solutions, whereas glute-

nins are polymers, consisting of high molecular

weight (HMW) and low molecular weight (LMW)

subunits, and these become extractable with aqueous

alcohol solutions only after the inter-chain (between

subunits) disulphide bonds are broken with reducing

reagents. Prolamin polypeptides differ greatly in the

content of cysteine residues, and can therefore be

classified into S-poor, S-rich, and HMW glutenin

subunits (Shewry et al. 1997). The limited S avail-

ability favours the synthesis of low-S gliadin storage

proteins and high molecular weight subunits of

glutenin at the expense of S-rich proteins (Wrigley

et al. 1980).

The composition of proteins is influenced sub-

stantially by the sulphur availability (Zhao et al.

1999, Naeem and MacRitchie 2003) because sul-

phur is a constituent of several essential compounds

such as cysteine, methionine, coenzymes, thioredox-

ine and sulfolipids (Singh 2003). Sulphur applica-

tion accelerates the metabolic pathway of protein

synthesis in the plant (Aulakh 2003), and serves to

increase the content of sulphur-bearing amino acids,

methionine and cysteine (Byers and Bolton 1979,

Wrigley et al. 1980, Aulakh 2003, Granvogl et al.

2008, Reinbold et al. 2008, Habtegebrial and Singh

2009).

Several studies (Castle and Randall 1987,

MacRitchie and Gupta 1993, Wieser et al. 2004)

have indicated that sulphur deficiency affects the

amount and the proportions of different gluten

protein types in wheat flour. Sulphur deficiency

caused a significant increase in the amount of

S-free v-gliadins and moderately increased the

amount of S-poor high molecular weight (HMW)

glutenin subunits. Additionally, it has been shown

that sulphur deficiency causes low yield and poor

technological properties of wheat, the latter of which

results in dough that is less extensible and more

resistant to extension and in loaves of smaller volume

and poorer texture (MacRitchie and Gupta 1993).

The supply of sulphur to the plant is important for

the quantitative composition of gluten proteins and,

therefore, for the technological properties of wheat

flour (Randall and Wrigley 1986, Zhao et al. 1999,

Koehler et al. 2004).

The results of field trials conducted in England

showed that even though additions of sulphur

fertilizer did not affect the total content of crude

protein in wheat grain, they tended to increase the

amount of gel protein. The gel protein fraction

contains predominantly glutenins which are closely

linked to bread-making quality (Zhao et al., 1999).

In wheat protein, the essential amino acid in

greatest deficit is lysine (McDonald et al. 2002,

Shewry 2009). The decrease in the relative lysine

content of high protein grain results from propor-

tional increases in the lysine-poor gluten proteins

when excess nitrogen is available, for example, when

fertilizer is applied to increase grain yield and protein

content (Shewry 2009).

In an earlier investigation (Jarvan et al. 2011) we

observed a significant effect of nitrogen and sulphur

fertilization on the yield and yield components of

winter wheat in two locations with different soil and

climatic conditions in Estonia. Therefore, grain

samples from this earlier study were selected to

further explore the effects of nitrogen and sulphur

fertilization on several grain quality parameters such

as protein and wet gluten content, gluten index and

some essential amino acids content in grain and

protein of wheat.

For the present study we have set the following

hypothesis: the sulphur fertilization of winter wheat,

with increasing yields may decrease the protein

concentration in grain but improve the quality of

protein.

Materials and methods

The present investigation is based on the field

trials conducted in 2004, 2005, 2007 and 2008

on break-stony soil at Saku in Northern Estonia

(59818?N, 24839?E) and in 2005 and 2009 on

pseudopodzolic soil at Auksi in Southern Estonia

(58827? N, 25836?E). The agrochemical properties of

these soils have been characterized in detail in our

earlier study (Jarvan et al. 2011). Incidentally, the

content of water-soluble S (ISO 11048) at the

beginning of the growing season determined by

using ICP (wave length 181.975 nm) was the follow-

ing: at Saku S 8�10 mg kg�1, and at Auksi S 6�12

mg kg�1. In the previous autumn under wheat

sowing with the complex fertilizer the plant nutrients

were applied at the following rates: in 2007 and 2008

at Saku � N12 P26 K50 S15 kg ha�1; in 2009 at Auksi

� N12 P26 K75 S9 kg ha�1. In the field trials of 2004

and 2005, wheat was not given any mineral fertilizers

in the previous autumn, because the phosphorus and

potassium contents in soil were sufficient.

The trials were performed with winter wheat

(Triticum aestivum L.) variety ‘Lars’, except in 2009

when the variety ‘Ada’ was sown at Auksi. Red clover

as preceding crop and green manure to wheat was

grown in trials at Saku and, in 2005, at Auksi. In

2009 at Auksi, wheat was preceded by oilseed rape.

The effect of sulphur as a plant nutrient on wheat

grain quality was investigated on the nitrogen back-

ground of N 100 kg ha�1 that was applied broadcast

628 M. Jarvan et al.

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as solid topdressing divided into two portions: N

60 kg ha�1 at the beginning of tillering and N 40 kg

ha�1 at the end of tillering. As detailed in Jarvan

et al. (2011), the dates of fertilizer application within

years somewhat differed because due to weather

conditions the wheat plants passed the growth stages

at different times. Two fertilizer variants were

compared � N (control) and NS. In the N-treatment

ammonium nitrate at the rate N100 was used. In the

NS-treatment the same nitrogen rate was applied

with Axan or Axan Super. These granulated fertili-

zers contained N 27% (N-NO3 13.5% and N-NH4

13.5%) and water-soluble sulphate-S 2.7 or 3.7%.

In the NS-treatment sulphur (S) was given at the

rate 10�13.6 kg ha�1. All trials included also a non-

fertilized 0-treatment � a so-called field background.

The field trials were performed on 25 m2 trial plots

in four replications. Weather conditions during

the growing period in both locations are shown in

Table I.

At the maturity stage, wheat grain yields from trial

plots were harvested with a combine harvester,

dried, sorted and calculated to 86% dry matter.

The grain samples of 1 kg weight were taken from

the yields of trial variants in four replications. These

samples were presented in the laboratory, where the

content of crude protein and wet gluten, and gluten

index of wheat grain were determined. Later, for

each trial treatment the grain samples of four

replications were gathered, mixed carefully and the

average grain samples were taken for amino acids

analysis. In these samples the contents of lysine,

threonine, cysteine and methionine were determined

in three replications.

The quality analyses were performed in the plant

production laboratory of the Agricultural Re-

search Centre. Protein was determined according

to AOAC Official Method 2001.11. The procedure

is applicable to the determination of Kjeldahl N

using digestion block and steam distillation and

autotitration unit. A ground laboratory sample is

digested in H2SO4 at boiling point, using catalyst

K2SO4�CuSO4 in tablets form. Using a steam

distillation unit acid is neutralized with NaOH and

the liberated NH3 is distilled into boric acid solution

and titrated with standardized sulphuric acid to a

colorimetric endpoint.

Wet gluten content and quality were determined

according to ICC Standard No. 155:1994. Gluten

separated from whole wheat meal by Glutomatic

equipment is centrifuged to force wet gluten through

a specially constructed sieve under standardized

conditions. The total weight is defined as gluten

quantity. The percentage of wet gluten remaining

on the sieve after centrifugation is defined as the

gluten index.

The determination of amino acids was performed

according to the following methods: EVS-EN

ISO 13903:2005 and HPLC UV (Perkin Elmer LC

System). Cysteine and methionine are extracted with

an oxidation mixture: performic acid (HCO2OH)�phenole (C6H5OH), then lysine and threonine are

extracted with a hydrolysis mixture: hydrochloric

acid (HCl)�phenole (C6H5OH). The amino acids

are separated by reversed-phase high-performance

liquid chromatography (HPLC) with precolumn

derivatization (FOMOC-Cl, ADAM) and are de-

tected at 263 nm. Column: RP C18 (150�4 mm,

5 mm i.d.) at 248C; eluent A: acetate buffer 50 mM

pH�4.2; eluent B: acetonitrile; gradient elution,

eluted within 45 min.

All results were based on four or three replicates.

The means were calculated for each variant and

the Tukey�Kramer honestly significant difference

(HSD) test was used to determine the differences

Table I. Mean air temperature and precipitation for growing seasons 2004�2009 at Saku and Auksi, and long-term average (1922�2009)

at Jogeva.

At Saku At Auksi

Month 2004 2005 2007 2008 2005 2009 Long term average

Air temperature, 8CMay 9.5 9.7 10.8 8.8 10.7 11.5 10.2

June 12.4 13.0 15.1 13.2 14.7 13.9 14.4

July 15.7 17.2 16.2 15.3 18.5 17.1 16.7

01�15 August 16.4 15.9 17.3 14.3 16.3 15.7 15.3

01 May�15 August 13.5 14.0 14.8 12.9 15.0 14.5 14.2

Precipitation, mm

May 35 47 27 10 67 17 50

June 75 39 8 96 43 85 66

July 259 82 42 53 48 136 81

01�15 August 25 92 18 111 96 68 40

01 May�15 August 394 260 95 270 254 306 237

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between the means (JMP 5.0.1 software; SAS

Institute, Cary, NC).

Results and discussion

The results of field trials conducted in 2004�2008

on the break-stony soil at Saku had shown a great

difference in the quality of wheat grains. The

contents of protein and wet gluten in grain varied

significantly depending on trial years (Table II).

Comparing these parameters, for example, in the

case of nitrogen fertilizer treatment at the rate of N

100 kg ha�1 it was evident that in 2004 and 2005

the grain protein content was about 14%, whereas in

2007 and 2008 the protein content resulted only in

about 10.5%. Also the wet gluten contents of wheat

grain in 2004 and 2005 were significantly higher

than in 2007 and 2008.

Although Thomason et al. (2007) have concluded

that protein level is typically greater in wheat grown

under stress of water and temperature this conclu-

sion in the full extent is not applicable for the results

of field trials conducted in 2004�2008 at Saku.

Comparing the weather conditions of the years, it

became evident that the amounts of precipitation

during the main growth period, from May to the

middle of August when the wheat was harvested,

were as follows: in 2004: 394 mm, 2005: 260 mm,

2007: 95 mm, and in 2008: 270 mm. Therefore, the

growth period in 2005 and 2008 was with normal

precipitation, 2004 with high rainfall but 2007 was

extraordinary dry. The springs of 2004 and 2005

were with sufficient and regular precipitation which

favoured the uptake of fertilizer nutrients applied as

topdressing. The high availability of nitrogen en-

hances growth and biomass formation in wheat

Table II. The effect of N and NS fertilization on the yield and some biological quality parameters of winter wheat grains in the field trials on

the break-stony soil at Saku.

Year

Parameter/fertilizer treatment* 2004 2005 2007 2008 Average 2004�2008

Grain yield, t ha�1 (n �4)

Without fertilizer 3.15b 4.58c 3.47b 3.44c 3.66c

N 3.44b 5.08b 5.66a 7.20b 5.34b

NS 4.92a 5.88a 5.92a 9.26a 6.50a

Protein content, % (n �4)

Without fertilizer 10.6b 11.1c 7.9c 7.8b 9.4c

N 14.1a 13.9a 10.6a 10.4a 12.2a

NS 11.6b 13.4b 10.1b 10.2a 11.3b

Wet gluten content, % (n �4)

Without fertilizer 23.0b 21.9b 11.2b 11.7b 17.0b

N 31.9a 31.6a 22.8a 19.4a 26.4a

NS 25.1b 30.2a 21.0a 18.6a 23.7a

Gluten index, % (n �4)

Without fertilizer 77a 79a 98a 90a 86a

N 45b 32c 75b 79b 58c

NS 74a 51b 90a 80b 74a

Cystein, g kg�1 (n �3)

Without fertilizer 2.60a 2.35b 1.47b 1.25b 1.92a

N 2.34b 2.23b 1.74a 1.45a 1.94a

NS 2.53a 2.91a 1.76a 1.48ab 2.17a

Threonin, g kg�1 (n �3)

Without fertilizer 3.17a 2.82ab 3.09b 2.01b 2.77a

N 3.73a 2.74b 4.03a 2.43a 3.23a

NS 3.75a 3.40a 3.90a 2.41a 3.36a

Methionin, g kg�1 (n �3)

Without fertilizer 2.04b 1.77a 1.12b 0.95b 1.47ab

N 1.86c 1.26b 1.44a 1.11a 1.39b

NS 2.14a 1.98a 1.40ab 1.15a 1.67a

Lysin, g kg�1 (n �3)

Without fertilizer 4.88b 3.28a 2.56a 2.68b 3.35a

N 5.24a 3.13a 2.77a 3.17ab 3.58a

NS 4.66c 3.65a 2.53a 3.44a 3.57a

*Fertilizer treatments: N: ammonium nitrate at the rate N 60�40 kg ha�1; NS: Axan at the rate N60 S6�N40 S4 kg ha�1 in 2004 and

2005 or Axan Super at the rate N60 S 8.2�N40 S5.4 kg ha�1 in 2007 and 2008. n: number of replicates.

Different letters in the same column indicate significant difference at p B0.05.

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plants as well as the transfer of nitrogen in the form

of amino acids to the growing grains (Wrigley et al.

1980, Zorb et al. 2010). Developing wheat grains

require nitrogen and sulphur to synthesize storage

proteins. During generative growth, either the ex-

ternal medium or reserves in vegetative tissues must

provide nitrogen and sulphur for protein synthesis

in the grains (Fitzgerald et al. 1999). According to

Timms et al. (1981) and Grove et al. (2009)

fertilization regimes of high nitrogen without giving

any extra sulphur may induce a sulphur deficiency in

the grains that will highly influence their quality.

In our trials conducted in 2004 and 2005 at Saku,

the fertilization of winter wheat with nitrogen at the

rate of N 100 kg ha�1 had an insignificant effect on

the grain yield, but it significantly affected the

protein content. In these trials under the influence

of nitrogen, the protein content in wheat grain

increased as follows: in 2004, from 10.6 to 14.1,

i.e. by 33.0%, and in 2005, from 11.1 to 13.9, i.e.

by 25.2%. Also the wet gluten content rose from

23.0 to 31.9 and from 21.9 to 31.6, i.e. by 38.7 and

44.3%, respectively. However, at the same time the

one-sided fertilization with nitrogen significantly

decreased the quality of grain protein. The first

sign of protein quality degradation was a great

decline in gluten index value, which decreased

from 77 to 45, i.e. by 41.6%, and from 79 to 32,

i.e. by 59.5% as compared with the unfertilized

variant. There were also some significant changes

in the contents of some amino acids under the

influence of nitrogen application.

In 2007, there was a great lack of preci-

pitation during growth from May to the middle

of July. Therefore, the uptake and moving of

topdressed nutrients to the wheat plants were

inhibited; for this reason, the plants at the maturing

stage had nitrogen and sulphur available in too small

quantities to synthesize grain protein in sufficient

amounts. In that year, the grain protein content

in N-treatment reached only 10.6% and in NS-

treatment 10.1%. The wet gluten content in wheat

grain by these treatments was 22.8 and 21.0%,

respectively.

In the field trial of 2008, the protein and wet

gluten contents of wheat grain in treatments were

more or less on the same level as in 2007. Their low

content might be caused, at first, from weather

conditions in the growing season. In 2008, the spring

until to the middle of June was relatively dry, and the

applied fertilizers, probably, could not take a sig-

nificant effect. Besides, rainy and cool days prevailed

at the maturity stage of wheat which affected the

protein synthesis negatively. Another reason for low

protein content might be the protein dilution effect,

because much higher yields were reached in fertilized

treatments this year than in previous years. By

Thomason et al. (2007), wheat yield and protein

concentration are inversely related due to dilution

effects within the plant. Also in the study of Weber

et al. (2008) higher grain yields were correlated with

lower protein concentration. The authors consider

the dilution effect to be a possible reason for the

lower protein concentration.

The sulphur application at the rate of S 10 to

13.6 kg ha�1 on the nitrogen background of N 100

kg ha�1 increased the wheat grain yield in all trials

conducted in 2004�2008 on break-stony soil at

Saku. As an average of four years, the grain yield

increased by 1.16 t ha�1, i.e. 21.7%. At the same

time, with the increasing yields the protein concen-

tration in grains decreased significantly in 2004,

2005 and 2007. As an average of these three trials,

the protein concentration decreased by 9.1%. Also,

the sulphur application tended to decrease the wet

gluten content; a significant decrease (21.3%) was

revealed in the trial conditions of 2004.

In trials conducted in 2005 and 2009 on the

pseudopodzolic soil at Auksi (Table III), the applica-

tion of nitrogen fertilizer at the rate of N 100 kg

ha�1 increased the winter wheat yield by 81.7 and

57.6% as compared with the unfertilized treatment.

The nitrogen application had a significant effect on

the quality of wheat grains because in both years the

contents of protein and wet gluten were increased.

The application of sulphur in addition to nitrogen on

pseudopodzolic soil did not have any positive effect

either on grain yield or on protein and wet gluten

Table III. The effect of N and NS fertilization on the yield and

some quality parameters of winter wheat grains in the field trials

on the pseudopodzolic soil at Auksi (n �4).

Year/fertilizer

treatment*

Yield,

t ha�1

Protein,

%

Wet

gluten, %

Gluten

index, %

2005

Without fertilizer 3.38b 11.5b 20.8b 86a

N 6.14a 13.2a 27.6a 61b

NS 6.63a 13.7a 29.3a 82a

2009

Without fertilizer 5.43b 11.8b 22.9b 86a

N 8.56a 12.6a 27.0a 77a

NS 8.65a 12.4ab 26.3ab 80a

Average 2005 and 2009

Without fertilizer 4.40b 11.5b 21.8b 86a

N 7.35a 12.9a 27.3a 69c

NS 7.64a 13.1a 27.8a 81b

*Fertilizer treatments: N: ammonium nitrate at the rate N 60�40 kg ha�1; NS: Axan at the rate N60 S6�N40 S4 kg ha�1 in

2005 or Axan Super at the rate N60 S 8.2�N40 S5.4 kg ha�1 in

2009.

n: number of replicates.

Different letters in the same column indicate significant difference

at pB0.05.

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content. The gluten index remained practically

unaffected by treatments, except in 2005 when in

N-treatment the gluten index value decreased by

29.1% as compared with the unfertilized variant or

by 25.6% when compared with the NS-treatment.

It is generally accepted that the values of the

gluten index range 60�90% in trade for bread-

making (Tayyar 2010). It has been found that

the optimum gluten index values for central

European wheat cultivars range between 75 and

90% (Curic et al. 2001).

In our research the results on gluten index of

wheat grain have given evidence for a great variation,

depending on the year and the treatment, from 32%

to 98% (Table II). The lowest gluten indexes were

found in 2004 and 2005 in treatment whereby

nitrogen fertilizer at the rate N 100 kg ha�1 without

sulphur was applied. Although the contents of

protein and wet gluten in wheat grain increased

considerably under the influence of nitrogen, the

gluten index values reached only 45% and 32%,

which cannot be regarded as sufficient for good

bread-making quality of wheat. As our further

research (Jarvan et al. 2006) with wheat flours

from different treatments had shown, several para-

meters of baking quality (stability and quality

number of dough, loaf ’s volume, height to diameter,

and porosity) decreased significantly when the gluten

index of wheat grain was below 50%. In comparison

with the unfertilized treatment the fertilization

with nitrogen at the rate N 100 kg ha�1 decreased

considerably the gluten index value of wheat grain

grown on the break-stony soil at Saku in 2004�2008

(Table II) and on the pseudopodzolic soil at Auksi in

2005 (Table III). Also the investigations of Garrido-

Lestache et al. (2004) revealed that increased nitro-

gen rates had a negative effect on the gluten index of

wheat. In another study, Ames et al. (2003) found

out that increased protein content as a result of

nitrogen fertilization has no significant influence on

gluten strength as measured by gluten index.

Compared with N-fertilized treatment, sulphur

supply at the rate S 10 or 13.6 kg ha�1 increased

significantly the gluten index of wheat, which rose

by 27.6% as an average of four year trials conducted

at Saku. In the field trials conducted at Auksi, the

application of sulphur increased the gluten index

of wheat grain by 34.4% in weather conditions of

2005, but had no effect in 2009. Also investigations

conducted in Spain by Garrido-Lestache et al.

(2004) have shown that sulphur application had no

significant effect on the protein quality indices of

wheat.

The biological quality of wheat as feed and as

food is highly dependent on the content and

composition of protein. With increasing protein

content, the percentage of some essential amino

acids (lysine, threonine and cystine among them)

in wheat grain will generally decrease (Maner 1987).

Of the 20 amino acids commonly present in proteins,

10 can be considered to be essential in that they

cannot be synthesized by animals and must be

provided in the diet (Shewry 2009). In wheat

protein, the essential amino acid in greatest deficit

is lysine (McDonald et al. 2002, Shewry 2009).

Lysine is very important in feeding animals and

poultry. For pigs, lysine has been shown to be the

first limiting amino acid, followed by threonine,

valine and methionine (Maner 1987).

The amino acid composition of wheat grain is

susceptible to fertilizer treatments (Byers and Bolton

1979, Mortensen et al. 1992, Zhao et al. 1999,

Granvogl et al. 2008, Zorb et al. 2010).

Byers and Bolton (1979) showed that sulphur

deficiency decreased the concentration of cysteine

and methionine (expressed as a percentage of total

recovered amino acids) in grain or flour markedly,

with the effect being more pronounced on cysteine

than on methionine. Because the methionine is not

the most limiting essential amino acid in wheat, the

effect of sulphur availability on the nutritional

value of wheat proteins would be less important if

the concentrations of other more limiting amino

acids, such as lysine and threonine, were not affected

(Byers and Bolton 1979). However, they also found

that both lysine and threonine were decreased as a

result of sulphur deficiency, particularly when the

nitrogen supply was high. Wrigley et al. (1980)

found that sulphur deficiency had a minor effect

on lysine concentration, but on the other hand it

caused a noticeable decrease in the concentration of

threonine.

In our studies conducted on break-stony soil in

2004�2008, it became evident that the application

of nitrogen and sulphur had a different effect

on some amino acids content in winter wheat grain

(Table II). In 2004, the fertilization with nitrogen at

the rate of N 100 kg ha�1 increased the lysine

content by 7.4%, but decreased the cysteine and

methionine content by 10.0 and 8.8%, respectively.

Both of these sulphur-containing amino acids are

very important for the development of good bread-

making quality of wheat flour (Timms et al. 1981).

According to Tea et al. (2007), the current practice

of applying large amounts of nitrogen fertilizers to

cereal crops without considering sulphur require-

ments is becoming a concern for crop quality. In

2004, under the influence of sulphur added at the

rate of S 10 kg ha�1 to nitrogen, the cysteine and

methionine contents increased by 8.1 and 15.1%,

respectively. In 2005, the differences in amino acid

contents depending on fertilizer treatments were

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more notable than in 2004. A major decline

(28.8%) in methionine content in the case of one-

sided nitrogen supply was observed. Under the

influence of sulphur applied to nitrogen, the con-

tent of several amino acids increased significantly as

follows: cysteine � 30.5%, methionine � 57.1%,

and threonine � 24.1%. In this respect, our results

are analogous to the findings of several other

researchers (Aulakh 2003, Koehler et al. 2004,

Granvogl et al. 2008) who have asserted that the

applied sulphur increased the sulphur-bearing

amino acids methionine and cysteine. Also, the

investigation of Habtegebrial and Singh (2009)

had shown a significant positive effect of

sulphur fertilization on both cysteine and methio-

nine content in wheat grain, and, in addition,

applied sulphur also improved the nitrogen use

efficiency.

In 2007 and 2008 when the weather conditions

were not favourable for protein synthesis, the ferti-

lizer application practically did not affect the content

of amino acids in wheat grain. Due to very different

weather conditions in 2004�2008, the values of the

amino acid contents varied highly, therefore their

four-years’ average values do not shown significant

differences. An exception was in the case of methio-

nine in which content in wheat grain increased under

applied sulphur by 20.1% as an average of four-year

trials.

In trials conducted in 2005 and 2009 on the

pseudopodzolic soil, the nitrogen fertilization at the

rate of N 100 kg ha�1 in both years significantly

increased the content of all amino acids determined

(Table V). In 2005, sulphur applied with nitrogen at

the rate of S 10 kg ha�1 increased the content of

sulphur-containing amino acids in wheat grain as

follows: cysteine 12.7%, and methionine 8.5%. In

the weather conditions of 2009, sulphur did not

increase the amino acid content in wheat grain.

Although in the field trials conducted on break-

stony soil at Saku the sulphur application � as com-

pared with the one-sided nitrogen fertilization � in

many cases significantly decreased the protein and

wet gluten content in wheat grain, it improved

the biological quality of protein. Recalculating the

concentrations of amino acids in wheat grain on

its content in wheat protein it became evident that

in the case of N-treatment the concentrations of

all amino acids in protein were lower than without

fertilizer (Table IV). This result coincides with

the results of other researchers (Timms et al.

1981, Shewry 2009, Zorb et al. 2010) stating a

loss of nutritional and bread-making quality of

wheat protein when excess nitrogen without sulphur

is available.

When comparing the amino acid concentra-

tions recalculated in wheat protein of N- and NS-

treatments, it appeared that the sulphur application

might increase the amino acids content as an average

of four years as follows: methionine 28.1%, cysteine

20.4%, threonine 11.1% and lysine 8.2%. From

the viewpoint of animal feeding, how much protein

and essential amino acids can be obtained from a

unit area is very important. Regardless of the

Table IV. The effect of N and NS fertilization on the amino acid content in the protein of winter wheat grown on the break-stony soil

at Saku.

Year

Amino acids in wheat protein/fertilizer treatment* 2004 2005 2007 2008 Average 2004�2008

Cystein, g kg�1

Without fertilizer 24.5 21.2 18.6 16.2 20.1

N 16.6 16.0 16.4 13.9 15.7

NS 21.8 21.7 17.4 14.6 18.9

Threonin, g kg�1

Without fertilizer 29.9 25.4 39.1 25.8 30.0

N 26.7 19.7 38.0 23.4 27.0

NS 32.3 25.4 38.6 23.6 30.0

Methionin, g kg�1

Without fertilizer 19.2 15.9 14.2 12.2 15.4

N 13.2 9.1 13.6 10.7 11.4

NS 18.4 14.8 13.9 11.3 14.6

Lysin, g kg�1

Without fertilizer 46.0 29.5 32.4 34.5 35.6

N 37.2 22.5 26.1 30.5 29.1

NS 40.2 27.2 25.0 33.7 31.5

*Fertilizer treatments: N: ammonium nitrate at the rate N 60�40 kg ha-1; NS: Axan at the rate N60 S6�N40 S4 kg ha-1 in 2004 and

2005 or Axan Super at the rate N60 S 8.2�N40 S5.4 kg ha�1 in 2007 and 2008.

The content and quality of protein in winter wheat grains 633

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decreased protein content in wheat grain, but thanks

to the increased yields, the application of sulphur

allowed an extra income of 83.0 kg protein to be

obtained from a hectare area. The total amounts

of amino acids in the yield from a hectare area

under the influence of sulphur were much higher

than without sulphur. As an average of four years,

the amino acids’ yields calculated on unit area

(kg ha�1) increased under influence of sulphur as

follows: methionine 46.4%, cysteine 36.1%, threo-

nine 21.8% and lysine 21.3%.

In conclusion, the hypothesis which was set up at

the start of the present study proved correct. On the

break-stony soil in 2004�2008, the sulphur appli-

cation at the rate of S 10 to 13.6 kg ha�1 on the

nitrogen background of N 100 kg ha�1 increased the

winter wheat yield by 1.16 t ha�1 on average, i.e. by

21.7%. With increasing yields the protein and wet

gluten concentrations in grain decreased significantly

but the biological quality of protein improved under

the influence of sulphur because the gluten index

value and the contents of amino acids in protein

increased. On the pseudopodzolic soil, the applica-

tion of sulphur in addition to nitrogen did not have

any positive effect either on grain yield or on protein

and wet gluten content. However, in weather condi-

tions of 2005, fertilization with sulphur increased the

content of sulphur-containing amino acids cysteine

and methionine in wheat grain.

Acknowledgements

Financial support from the Estonian Ministry of

Agriculture through the project ‘Improving the food

and feed quality of cereals, grain legumes and oil crops

by implementing economically effective and environ-

mentally sustainable agrotechnical methods’ (2006�2010) is much appreciated. We thank Mr Jaanus

Rebane and Mrs Ann Akk for the description of

analysis methods. We thank also the staff of the plant

production laboratory of the Agricultural Research

Centre for their accurate analytical work. The authors

are grateful to Mrs Helena Parenson for her linguistic

consultations.

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