1. Introduction
Wheat protein content and baking quality, highly depend on genotypic and environmental
factors and it is recognised that variation in protein content and composition significantly
modify wheat quality. Rousset et al., 1985; Bietz, 1988; Borghi et al., 1995.
Many studies have been conducted in an at- tempt to explain wheat quality variation as a
function of genetic variation in protein composi- tion. The major storage proteins in wheat are the
gliadins and glutenins. Gliadins are subdivided into a, b, g and v units and glutenins into low
and
high molecular
weight LMW-GS
and HMW-GS. The genetic control and the relation-
ships between gluten protein composition and quality characteristics are better and better under-
stood Shewry et al., 1989; Colot, 1990; Halford et al., 1992; Gupta and MacRitchie, 1994;
Popineau et al., 1994; Weegels et al., 1996; Fido et al., 1997; Martinant et al., 1998; Metakovsky and
Branlard, 1998.
With regard to environmental effects several previous studies reported, at different levels of
investigation, some
variations in
protein composition:
the amino-acids composition g aa100 g proteins varies with the N content of the grain
Mosse et al., 1985; Triboi et al., 1990; Bulman et al., 1994; Boila et al., 1995;
the quantity of N and S in the grain increases with N content, but the NS ratio decreases:
this reflects a change in the ratio between protein with lower sulphur content such as
certain gliadins and higher sulphur proteins such as glutenins Byers and Bolton, 1979;
Triboi et al., 1990; MacRitchie and Gupta, 1993;
the gliadinglutenin ratio increases with the quantity of N in the grain Terce-Laforque and
Pernollet, 1982; Triboi et al., 1990; Triboi and Leblevenec, 1995;
the concentration of some gliadin bands, mea- sured by electrophoresis and densitometry,
changes with the variation in N supply to the grain Brandlard and Triboı¨, 1983.
Progress in analytical technology and particu- larly in RP- and SE-HPLC has enabled a better
understanding of the effects of protein composi- tion Marchylo et al., 1989; Lookhart, 1997. As
electrophoresis, HPLC not only separates differ- ent gliadin and glutenin subunits but also
quantifies individual or pooled subunits. How- ever, this technique has been used in few studies
and quantitative determination of protein units and subunits and their relationships with quality
have seldom been described Marchylo et al., 1989; Seilmeier et al., 1990; Sutton et al., 1990;
Kolster et al., 1991; Sutton, 1991; Wiesser et al., 1994. Subsequently, the quantitative changes can
be accompanied by qualitative changes reflected in the ratios between different components Triboi
and Triboı¨-Blondel, 1998; Daniel et al., 1998a,b. Changes in the ratios between different protein
units and subunits have rarely been signalled, and the effects on the technological properties have
often not been established.
Generally, two aspects can be considered in the research on environmental effects:
1. the change in grain composition and particu- larly in the quantity and quality of proteins;
2. the relationship between protein changes and quality parameters.
This paper deals with the first aspect: the effect of environmental conditions N fertilisation rate,
site and growing season on the quantitative and qualitative variation of wheat glutenin and gliadin
units and subunits. A new method of sequential extraction of proteins followed by quantitative
determination by RP-HPLC was performed in order to analyse the qualitative changes induced
by quantitative variations and to establish the relationships between different protein units and
subunits for modelling purposes.
2. Materials and methods
2
.
1
. Field experiments Bancal and Rinconada bread wheat varieties
were grown in irrigated plots of Typic Xerofluvent soils at two sites of the Ebro Valley Torregrossa
and Bell.lloc; Spain during two growing seasons
1994 – 1995 and 1995 – 1996. The soil at Bell.lloc was a deep silty-clay-loam with a high N content
and 2.9 organic matter. The soil at Torregrossa was a sandy-loam with a low N content and 1.9
organic matter. Surface irrigation was applied from stem extension stage 31 Zadoks until the
formation of kernels Zadoks 70, three times during the growing season with an approximate
total of 2000 m
3
h. Rinconada and Bancal are alternative types of wheat without need for ver-
nalisation. The HMW-GS composition is 1, 7 + 8, 5 + 10 for Rinconada and 7 + 9, 5 + 10 for
Bancal.
The experimental design was a split-plot ran- domised complete-block with four replicates. The
main plots consisted of wheat cultivars, while the subplots were subjected to three N fertilisation
treatments, 0, 100 and 200 kgha. Only two field replicates were used in this study, hence 48 sam-
ples two replicates, three N, two varieties, two sites, two years were analysed for protein
composition.
2
.
2
. Sequential extraction of protein The sequential extraction of protein of whole
grain flour was used Marion et al., 1994; Nicolas et al., 1997. Albumin-globulin was extracted
from 833 mg of ground wheat with 25 ml buffer A 0.05 M sodium phosphate pH 7.8, 0.05 M NaCl
for 1 h at 4°C. Samples were centrifuged at 18 000 rpm for 20 min at 4°C. Amphyphilic proteins
were extracted from the previous residue with 25 ml of 2 wv Triton X-114 in buffer A, for 1 h
at 4°C. After centrifugation 18 000 rpm for 20 min at 4°C, gliadin was extracted from the
residue with 25 ml of 70 vv aqueous ethanol for 1 h at 20°C. To obtain glutenin, after centrifu-
gation the residue was extracted overnight with 25 ml of buffer B 0.05 M di-sodium tetraborate pH
8.5, 2 vv b-mercaptoethanol, 8 M urea and 1 gl glicyne at 20°C. Samples were centrifuged at
18 000 rpm for 20 min. An amount of 3.5 ml of
the supernatant
was alkylated
with 4-
vinylpyridine. After alkylation, 3.5 ml of 2- propanol was used to precipitate polysaccharides.
Samples were centrifuged at 18 000 rpm for 20 min. Glutenin was in the supernatant.
2
.
3
. RP-HPLC of gliadin and glutenin The gliadin and glutenin composition of the
supernatants was analysed by RP-HPLC Kontron 422425 system, with a Nucleosil C18 column 300
A , , 5 mm, 30 nm, 250×4.6 mm at 50°C with a
C18 gard column 300 A , , 410 mm.
Two eluants were used: A water containing 60 vv acetonitrile and 0.07 vv trifl-
uoroacetic acid TFA; B water containing 0.1 vv TFA.
For the gliadin analysis the gradient was: 0 – 1.22 min — 41.7 A, 8 min — 46.2 A, 65
min — 83.0 A, 69 min — 100 A. The glutenin gradient was: 0 – 6 min — 41.7 A; 46
min — 66.7 A; 66 min — 91.7 A; 67 min — 100 A. For both analyses, the flow rate was
1.0 mlmin and the injection volume was 75 ml. Detection was at 220 nm with Shimadzu SPD6A.
Each class of glutenin was quantified by inte- gration of chromatogram areas, between 29 and
36 of acetonitrile for HMW-GS and between 36 and 51 for LMW-GS. Kontron data system
450-MT2 software was used to integrate the chro- matograms, which were standardised and whose
peaks were numbered as shown in Figs. 1 and 2. For each subunit and peak, protein content was
expressed as the amount area, mVmin per mg of flour or as a proportion of the total gliadin
and glutenin unit and subunit area. Total glutenin and gliadin were calculated as the total chro-
matogram area mVmin per mg of flour.
3. Results
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