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
3
.
1
. Grain weight GW
, protein content PC
and quantity per grain QNG
GW have a coefficient of variation CV of 8 Table 1. N fertilisation N was the main factor
of variation. An increase in N fertilisation rate significantly reduced grain weight from 47.3 mg
grain without N to 43.5 mggrain. Year Y and site S did not significantly affect grain weight,
but
the interactions
YS and
YN were
significant.
E .
Triboi et
al .
Europ .
J .
Agronomy
13 2000
47 –
64
Table 1 Environmental effect on grain weight, total and storage protein content
a
Grain weight Protein
Gliadin area
b
Glutenin area
b
Ratios Mg Ngrain
mg Gliadin protein
Gluteninprotein Gliadinglutenin
Variety V
236 a 15.0 a
16.2 a 46.2 a
0.94 a 1189 a
Bancal 14.7 a
223 a 293 b
13.7 b 18.8 b
Rinconada 0.74 b
44.3 a 1195 a
15.5 b 216 a
Site S
291 a 15.6 a
17.9 a 44.5 a
0.89 a 1278 a
Bell.lloc 16.2 a
254 a 185 b
241 b 13.1 b
17.1 a 0.80 b
45.7 a 1107 b
13.9 b Torregrossa
Year Y
15.2 a 215 a
248 a 13.9 a
16.0 a 0.87 a
44.5 a 1995
1171 a 283 b
14.8 a 18.9 b
0.81 a 224 a
46.2 a 1996
15.0 a 1214 a
N kgNha
230 a 12.9 a
16.0 a 0.83 a
47.3 a 1172 a
14.7 a 187 a
269 b 14.8 a
17.8 ab 0.86 a
225 b 100
14.9 a 1180 a
45.3 ab 246 b
299 b 12.9 a
18.6 b 0.84 a
43.5 b 1225 a
16.1 b 200
Significance of single
interactions SY
SN YN
SV YV
NV 8.9
8.1 8.7
13.3 9.6
8.0 3.3
10.7 CV
a
Means in the same column followed by the same letter are not significantly different are PB0.05. Significant at PB0.05.
Significant at 0.01.
b
Area is given in mVmin mgflour. CV, coefficient of variation.
Variation in the quantity of N per grain QNG, mg Ngrain was higher than that of grain weight
CV, 10.7. The QNG depended significantly on the site, with an average of 1278 mggrain at
Bell.lloc and 1107 mg at Torregrossa, confirming a higher soil N supply at Bell.lloc. However, there
was a significant interaction between site and N fertilisation, possibly resulting from the higher
soil N content at Bell.lloc that reduced the re- sponse of N grain content to N fertilisation. De-
Fig. 1. RP-HPLC of Bancal and Rinconada glutenin. Numbers in chromatogram are peak designation. Peaks eluted before 45 min are HMW, while those eluted after are LMW-glutenin.
Fig. 2. RP-HPLC of Bancal and Rinconada gliadin. Numbers in chromatogram are peak designation.
spite the fact that the most important factors of variation were different for the two variables site
for QNG and nitrogen for GW, they were not totally independent because the coefficient of vari-
ation of their ratio, i.e. protein content PC, was smaller only 3.3 Table 1. PC was mainly
affected by site and N fertilisation 63 of total variation. Wheat variety accounted only for 4
of total variation and growing season did not significantly affect PC. The effect of N fertilisa-
tion on PC differed between sites since there was no response in Bell.lloc, possibly due to its ini-
tially high soil N contents data not shown and where the average protein content was already
very high in the absence of N fertilisation, i.e. 16.4.
3
.
2
. Gliadin and glutenin content in the flour and in total protein
Gliadin content in the flour was affected only by site + 37 at Bell.lloc and by N fertilisation
+ 32 at 200 N but not by variety and year Table 1. These two factors explained 52 of the
total variance. Moreover, a significant interaction between site and N fertilisation was observed. At
Torregrossa, gliadin content increased by about 80 as a consequence of N fertilisation whereas
at Bell.lloc the increase was of about 20.
In contrast, glutenin content was significantly affected not only by site + 21 at Bell.lloc and
N fertilisation + 30 at 200 N but also by year + 14
in 1996
and variety
+ 24 in
Rinconada. Several significant interactions be- tween factors were obtained Table 1. At Torre-
grossa, glutenin content was higher in 1995 – 1996 than in 1994 – 1995, whereas no differences were
observed at Bell.lloc between glutenin content in the two growing seasons SY interaction. In
Rinconada, wheat glutenin content was higher in 1994 – 1995 than in 1995 – 1996, whereas no differ-
ences were observed at Bancal. YV interaction.
Despite the large variation in gliadin and glutenin contents in flour, their ratio was rela-
tively constant. The variety was the main factor of variation: the mean of this ratio was 0.94 for
Bancal and 0.74 for Rinconada. This is in agree- ment with several studies Kolster et al., 1991;
Pechanek et al., 1997. However there are also a site effect Bell.loc 0.89, Torregrossa 0.80 but
smaller that variety.
Concerning protein composition, variety and N fertilisation were the main factors affecting both
the gliadin to total protein and glutenin to total protein ratios. The year, alone or in interaction
with site or variety, was significant only for the glutenin content in total protein. Thus, glutenin
seems to be more environmentally dependent than gliadin.
All the modifications in gliadin and glutenin contents of flour were related to total protein
content. The coefficients of determination were higher for gliadin than for glutenin Fig. 3. This
is in agreement with previous studies Gupta et al., 1992, although other authors Andrews et al.,
1994 reported a better correlation with glutenin.
To take into consideration only nitrogen accu- mulation and to eliminate the effect of C accumu-
lation on N percentage, we calculated the quantity of gliadin, glutenin and total protein per grain.
Fig. 3. Amount of gliadin A and glutenins in flour B as a function of grain protein content in two bread wheat
varieties Bandal and Rinconada. Data consist of means of two sites Torregrossa and Bell.lloc, 2 years 1994 – 1995 and
1995 – 1996 and three N-fertilization rates 0, 100 and 200 kg Nha. Units of area are mVmin. The number of observations
was 24 for each regression.
Fig. 4. Amount of gliadins A and glutenins in flour B as a function of grain N content mg Ngrain in two bread wheat
varieties Bancal and Rinconada. Data consist of means of two sites Torregrossa and Bell.lloc, 2 years 1994 – 1995 and
1995 – 1996 and three N fertilisation rates 0, 100 and 200 kg Nha. Units of area are mVmin. The number of observations
was 24 for each regression.
protein content, the percentage of glutenin did not. In the present study, both the proportion of
gliadin and glutenin in total protein increased with increasing total protein content R
2
, 0.64 and R
2
, 0.40 P B 0.01 respectively, data not shown. The increase in the proportion of gliadin and
glutenin was probably related to the decrease in the non-storage protein fraction as shown by
Gupta et al., 1992, Jia et al., 1996b. Finally, the gliadin to glutenin ratio, reflecting a qualita-
tive change in protein composition, increased with N content per grain Fig. 5, suggesting that as N
accumulation increased, gliadin increased prefer- entially to glutenin.
3
.
3
. Analysis of glutenin composition Chromatograms of glutenin in Bancal A and
Rinconada B are presented in Fig. 1. The peak areas that were used to determine HMW- and
LMW-GS are indicated as well as the name given for every peak. HMW- and LMW-GS content
and the HMWLMW-GS ratio are presented in Table 2.
Fig. 5. The gliadinglutenin ratio as a function of grain N content mg Ngrain in two bread wheat varieties Bancal and
Rinconada. Data consist of means of two sites Torregrossa and Bell.lloc, 2 years 1994 – 1995 and 1995 – 1996 and three
N fertilisation rates 0, 100 and 200 Kg Nha. The number of observations was 24 for each regression.
The N content of grain as well as protein con- tent also showed a significant linear relationship
with the gliadin and glutenin contents, however gliadin was better correlated with the N content
of grain than glutenin Fig. 4.
Protein composition, expressed as gliadin to total protein and glutenin to total protein ratios
also changed with protein content. Previous stud- ies Brandlard and Triboı¨, 1983; Gupta et al.,
1992 reported that while the percentage of gliadin in total protein increased with the flour
Table 2 Environmental effect on glutenin subunit content
a
LMW
b
HMWLMW HMWprotein
LMWprotein HMW
b
Variety V
85 a Bancal
154 a 0.55 a
5.7 a 10.4 a
191 b 0.56 a
6.5 b 102 b
12.3 b Rinconada
Site S
187 a 0.56 a
104 a 6.4 a
Bell.lloc 11.5 a
158 b Torregrossa
0.56 a 83 b
5.9 b 11.2 a
Year Y
157 a 0.61 a
5.9 a 10.1 a
1994–1995 91 a
188 b 0.51 a
96 a 6.4 b
1995–1996 12.5 b
N kg Nha 149 a
0.59 a 81 a
5.7 a 10.4 a
176 b 0.53 a
100 6.1 ab
92 a 11.7 b
193 b 0.56 a
107 b 6.6 b
200 11.9 b
Significance of single interactions SY
SN YN
SV YV
NV 9.7
11.9 9.9
CV 9.4
10.2
a
Means in the same column followed by the same letter are not significantly different are PB0.05. CV, coefficient of variation.
b
Area mVminmg flour of high HMW and low LMW molecular weight glutenin. Significant at PB0.05.
Significant at 0.01.
Like glutenin content, the HMW and LMW glutenin content in flour was significantly affected
by variety, site and N fertilisation. The year and its interaction with variety YV only had an
effect on LMW-GS. At Bell.lloc, where the soil had a high N content, N fertilisation increased the
quantity of the different subunits by about 12, whereas at Torregrossa the increase of each sub-
unit as a result of N fertilisation was important: 40 and 70 for HMW and LMW-GS respectively
SN interaction, data not shown.
The composition of total proteins was also modified. The HMW-GS to total protein ratio,
and the LMW-GS to total protein ratio changed significantly with N fertilisation, site, year and
variety Table 2. Some single interactions, partic- ularly with N fertilisation NS, NY, NV only
had a significant effect on LMW-GS content. The change in protein composition was also
reflected in the variation of the HMW- to LMW- GS ratio. The main factor of variation was N
fertilisation, alone or in interaction with year, site and variety. Furthermore, the analysis of variance
for this ratio showed a different pattern for the two varieties VY, VN interactions: contrary to
Bancal in Rinconada this ratio changed signifi- cantly with growing season and N fertilisation.
The variation in HMW- and LMW-GS content was related to total glutenin content: HMW- and
LMW-GS contents increased with flour glutenin content Fig. 6A; R
2
\ 0.84. The increase in
LMW-GS seemed to be greater than that in HMW-GS, and consequently the HMW- to
LMW-GS ratio be lower MacRitchie and Gupta, 1993. However, no significant trend in the
HMW- to LMW-GS ratio was observed Fig. 6B
3
.
3
.
1
. Composition of HMW-GS According to retention time by chromatogra-
phy column HMW-GS were divided into 11 peaks Fig. 1. These peaks were grouped into four
pools: 30 –31, 33–36, 37–40 and 41–44. Table 3 presents the average amount and percentage of
HMW-GS for each peak and the correlation co- efficients with total HMW-GS.
The data shown a genetic effect on HMW-GS composition. Although there were 11 peaks in the
two varieties and the name given for each peak was the same, there were slight differences in the
retention time of certain peaks 34, 39, 42. For example the retention times for Bancal and
Rinconada were 34.5 and 33.8 min for peak 34, 39.4 and 39.1 for peak 39 and 42.3 and 42.9 for
peak 42, respectively. In both varieties, peaks 34 and 39 were the peaks with the highest of HMW-
GS content. In Bancal, each of these peaks repre- sented about 25 of total HMW glutenin and in
Rinconada, peak 34 represented 18 and peak 39 represented 29 of total HMW glutenin.
Generally, in the two varieties the amount of individual HMW peaks increased significantly as
HMW-GS increased r \ 0.43. The three major peaks 30, 34 and 39 were those with the highest
correlation with the total HMW-glutenin area r \ 0.77. The analysis of the percentage of each
peak for total HMW glutenin, revealed a different trend in the two varieties. Although in both vari-
eties the total amount of individual peaks in- creased as HMW-GS increased, the proportion of
every peak with regard to total HMW-GS was different: only the proportion of one pool 41 –44
varied in Bancal wheat, whereas four varied in Rinconada. In the latter variety, the proportion of
the three major peaks increased with HMW-GS content, and peak 40 was significantly lower.
However, despite their statistical significance, the correlation coefficients of these relations were
very small r 0.45; Fig. 7.
3
.
3
.
2
. LMW composition LMW-GS were divided into 15 peaks in Bancal
and into 17 peaks in Rinconada Fig. 2. Some of these peaks were pooled and some were consid-
ered as individual peaks. Table 4 presents the retention times for different LMW-GS peaks, the
average amount and percentage of each peak, and the correlation coefficients with total LMW-
GS.
Some LMW-GS peaks were specific to the vari- ety: peaks 49 –7, 51–5 and 59 were present only in
Rinconada and peak 58 only in Bancal. In Bancal there were five peaks 49, 51, 52, 53 and 54 and
in Rinconada there were three peaks 51 –5, 52
Fig. 6. HMW and LMW-GS A and the HMW-LMW-GS ratio B as a function of glutenin protein in two bread wheat
varieties Bancal and Rinconada. Data consist of means of two sites Torresgrossa and Bell.lloc, 2 years 1994 – 1995 and
1995 – 1996 and three N fertilisation rates 0, 100 and 200 kg Nha. Units of area are mVmin. The number of observations
was 24 for each regression.
Table 3 Environmental effect on HMW-glutenin composition: RP-HPLC peak characteristics
Bancal Variety
Rinconada Amount
Peaks Percentage
Retention Retention
Percentage Amount
time area
a
time area
a
of HMW r with HMW
of HMW r with HMW
Pool
30
–
31
13.19 15.5
– 11.71
11.5 –
29.7 30
7.76 9.0
– 29.9
6.16 5.9
0.45 5.43
6.5 –
31.1 31.3
5.55 31
5.6 –
Pool
33
–
36
35.17 41.5
– 31.35
30.3 –
6.01 7.1
– 33.1
33 4.01
33.2 3.9
– 21.30
25.1 –
33.8 34.5
18.65 34
18.0 0.42
36.0 36
7.85 9.3
– 36.0
8.69 8.4
– 27.88
32.8 –
39.57 38.9
Pool
37
–
40
– 4.99
5.9 –
37.8 37.7
5.52 37
5.5 –
21.66 25.4
– 39.1
39 31.13
39.4 29.0
0.45 1.23
1.5 –
40.3 40.3
2.91 40
3.5 −
0.46 Pool
41
–
44
8.41 10.1
− 0.41
19.50 19.3
– 7.87
9.4 –
41.1 41.5
8.26 41
8.2 –
42.3 42
0.03 0.03
– 42.9
5.80 5.6
– 0.51
44 0.6
43.9 –
44.2 5.44
5.3 –
a
Area is given in mVmin. Statistical significant at PB0.05.
and 54 each of which represented more than 10 of total LMW glutenin.
Generally, in the two wheat varieties, the amount of individual LMW-GS peaks increased
significantly as LMW-GS increased r with LMW \ 0.54. Although the total amount of indi-
vidual peaks increased as LMW-GS increased, the percentage of peaks were more constant in Bancal
than in Rinconada. In the Bancal variety, only the percentage of peaks 51 and 58 –5 increased signifi-
cantly with LMW-GS, whereas in Rinconada the percentage of several peaks changed Fig. 8;
pools 45 –48 and 49–50, peaks 53–9 and 56 de- creased significantly as LMW-GS increased and
peaks 51, pool 51 –52 and peak 54 significantly increased Table 4. These results suggest that the
synthesis of certain LMW protein fractions in wheat cultivars also depends on the environment.
3
.
4
. Analysis of gliadins Gliadin fractions were divided into 23 peaks in
Bancal and into 25 peaks in Rinconada Fig. 2. Some of these peaks were grouped into different
pools and some were considered as individual peaks. Table 5 indicates the most important char-
acteristics of the different peaks: retention time, amount of gliadin, percentage of gliadin and cor-
relation with the total amount of gliadin.
Peaks 19, 21, 22, 37 and 55 were present in Rinconada and not in Bancal and peaks 33, 58,
58.5, 59 and 59 were present only in Bancal but in small quantity Table 5.
There were six peaks each of which represented more than 7 of total gliadin subunits: 42, 45, 47,
49, 53, 62 in Bancal and 42, 45, 48, 49, 53 and 54 in Rinconada. Other individual peaks represented
less than 7 of total gliadin.
Generally, in the two wheat varieties, the amount of individual peaks increased significantly
as gliadin increased r 0.80 – 0.90, but the per- centage of peaks relative to total gliadin showed a
different pattern Fig. 9. For both varieties, as gliadin increased the percentage of certain peaks
increased r \ 0 while that of certain others de- creased r B 0 Table 5. Previous research
Brandlard and Triboı¨, 1983 also showed a varia- tion in the proportion of gliadin subunits with
changes in total protein content.
4. Discussion
