Reprinted from Crop Science Vol. 34, No. 2

Changes in Protein Compl~xes of Durum Whe~t in :Developfog ~eed·~ Florence B6netrix, Fran~ois Kaan, and Jean-Claude Autran*

Changes in Protein Complexes of Durum Wheat in Developing Seed Florence Benetrix,

Fran~is

Kaan, and Jean-Claude Autran*

ABSTRACT

Glu-B3 locus, genetically linked with the Gli-Bl locus (Pogna et al., 1988). Recently, significant correlation was shown between pasta quality potential and the ratio of aggregative fractions, especially LMW subunits of glutenin, in the endosperm, whereas other aggregative fractions, such as HMW glutenin subunits, played a less significant role in the differences in gluten quality amongst durum wheat genotypes (Autran and Galterio, 1989). Durum wheats must be bred for high quality potential and stability of quality expression; however, 1 d tood d be redi ted the latter is poor Y un ers an cannot P c by breeders. To investigate mechanisms of expression of seed quality, a relevant approach is to study accumulation of storage proteins in the wheat kernel during grain development. Results in this field are contradictory, however, mainly because different techniques and extracting agents have been used. For instance, according to Reeves et al. (1986), all storage proteins are synthesized from 12 to 15 d post-anthesis. In contrast, Skerritt et al. (1988) reported a chronologica1 accumulation of gliadins, a few 1 ·cal days after the glutenin synthesis. Because techno ogi quality of wheats is primarily determined by the occurrence of large protein aggregates and the size and composition of these protein aggregates are influenced by environmental conditions (Kaczkowski et al., 1987), to only study protein monomers or reduced subunits by electrophoresis is unlikely to give insights into mechanisms of expression of quality during grain development. The recent introduction of SE-HPLC in the study of bread-wheat protein aggregates allowed more accurate determination of the size range of unreduced protein complexes than did solubility methods (Autran, 1994) and allowed identification of various indicators of baking quality (Dachkevitch and Autran, 1989). In the same way, Millet et al. (1991) showed that the distribution of protein aggregates in the barley (Hordeum vulgare L.) kernel was related to malting quality. · In the present study, SE-HPLC and SOS-PAGE were used to investigate changes in protein complexes to relate expression of quality with mechanisms of protein aggregation. Analyses were carried out on kernels sampled during grain development from two durum wheat cultivars, each representing a quality type.

End-use quality of wheat derives from the functional properties of its storage proteins. Storage proteins are synthesiud during grain development and undergo modifications mainly during grain dehydration, with increased level of aggregation. This study was conducted to determine whether changes In protein complexes of durum wheat [Tritieum turgidum (Desf.)J during seed development relate to seed quality. Protein extracts of developing seeds of cultivars Capdur and Tomdair were reduced and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SOS-PAGE). Results conftrmed synthesis of both gliadins and glutenin subunits early during maturation with qualitative compositions remaining nearly constant. Sizeexclusion high-performance liquid chromatography (SE-llPLC) on unreduced protein extracts was used to follow quantitative changes in distribution of protein complexes. Among the five chromatographic fractions corresponding to diflerent sizes of aggregates or monomers, Ft (excluded peak) and F2 (complexes of Intermediate size) increased more rapidly in Capdur (good pasta quality) than in Tomclair (poor pasta quality). Cbaracteri7.atlon of the chromatographic fractions by electrophoresis showed that low-molecular-weight (LMW) subunits of glutenln are mainly involved in the largest complexes and are the ones most dearly showing differences between cultlvars. The tendency or LMW subunits to aggregate during grain dehydration may help explain differences round in puts quality among durum wheat cultivars.

D

is widely considered the best wheat for pasta products because of its excellent amber color and superior cooking quality. Differences in cooking quality among wheats are attributed to the protein content and composition of the grain endosperm (Feillet, 1977; 1988). Although the classical Osborne's scheme (Osborne, 1907) for protein fractionation continues to be widely used, other approaches have been suggested. For instance, Miftin et al. (1983) and Shewry et al. (1984) proposed a nomenclature related to functional properties of wheat storage proteins, including S-poor proteins (rogliadins), S-rich proteins (a-, P-, and y-gliadins), and LMW and high-molecular-weight (HMW) subunits of glutenin. A major breakthrough in understanding the genetic basis of durum wheat quality was realized by Damidaux et al. (1978) with the discovery of a relationship between the electrophoretic pattern of y-gliadins and gluten strength. Allelic type y-45 was associated with a strong gluten, whereas allelic type y-42 was associated with a weak gluten. In fact, the positive effect of y-gliadin 45 originated from aggregative subunits of glutenin of the URUM WHEAT

MATERIALS AND METHODS Wheat Samples. The two French durum wheat cultivars selected ·were Capdur, an excellent pasta-making-quality wheat, and Tomclair, a very poor pasta-making-quality wheat.

F. Ben6trix and J.C. Autran, Lab. de Technologie des C~reates, INRA, 2 Place Viala, 34060 Montpellier Cedex, France; and F. Kaan, Station d'Am6lioration des Plantes, INRA, 2 Place Viala, 34060 Montpellier Cedex, France. Received 8 Feb. 1993. *Corresponding author.

Abbreviations: SOS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SE-HPLC, size-exclusion high performance liquid chromatography; LMW, low molecular weight; HMW, high molecular weight; Fs, soluble fraction; Fi, insoluble fraction; cbnt, daily mean temperature.

Published in Crop Sci. 34:462-468 (1994).

462

BENETRIX ET AL.:

DURUM WHEAT PROTEIN COMPLEX CHANGES

.. Plants were grown in 1989 at the Montpellie.r INRA experimental field (Montpellier, France). At anthes1s (10 to 16 May), heads were labelled and then sampled at 2- to 3-d intervals until fully ripe. Nighttime and daytime temperature ranges were recorded daily. Maximum temperatures ranged from 17 .3 to. 27 .9 °C in May and from 20.3 to 34.0 °C in June. Excised heads were frozen at -20 °C, freeze-dried, and threshed. Dry seeds were finely ground in a Tecator Cyclotec laboratory mill (Tripette et Renaud, Paris) and stored at 4 °C. Because temperature affects the formation of grain storage components, results are reported as a function of the cumulative daily mean temperature after flowering (Cerning and Guilbot, 1973). Protein Extraction. To be analyzed by SE-HPLC, proteins were extracted from ground seeds (80 mg) by 10 mL of 0.1 M sodium phosphate buffer (pH 6.9) containing 2% sodium dodecyl sulfate (SDS) for 2 hat 60 °C. Extractions were followed by centrifugation for 30 min at 37 500 x g at 20 °C. The amount of proteins extractable by the phosphate-SOS buffer was defined as the soluble fraction (Fs), expressed on a percentage of total protein basis ..The percenta~e of insolu~le fraction (Fi) was determined by KJeldahl analysis of the residues (%Fs + %Fi = total protein content) as in Dachkevitch (1989). SE-llPLC. Instrumentation for SE-HPLC was described previously (Dachkevitch and Autran, 1989). A Beckman (Carlsbad, CA) TSK4Q09SW size-exclusion analytical column (7.5 by 300 mm, 450 A) protected by a guard column (7.5 by 75 mm, 250 A) was used. A 0.1 M sodium phosph~te buffer (pH 6.9) containing 0.1 % SDS was used as eluent with a flow rate of 0. 7 mL/min. Twenty microliters of supernatant were applied to the column using an automated sample injector. The column e1Huent was monitored at 214 run and 0.1 absorbance units full scale. The chromatograms were analyzed through Nelson analytical software, which permitted integration of the elution curve (Fig. 1). The total area under the elution curve, corresponding to the soluble fraction Fs, was divided into five main peaks representing different sizes of aggregates and monomers. Thyroglobulin (mol. wt. 669 kDa), alcohol dehydrogenase (mol. wt. 150 kDa), and bo~µte serum albumin (mol. wt. 66 kDa) were used as molecular weight

66.000

12.000

+ + 669,000

+

standards. Percentages of fractions were calculated by integrating data between the following molecular weight limits:

Fl> 600000 F2 = 100 000 to 600 000 F3 = 55000 to 100000 F4 = 20 000 to 55 000

F5

< 20000

All quantitative data are only expressed on a relative basis (i.e., in percentages of total area ~f c.hro~atograms). Altho~gh expressing data on an absolute ~as1s (1.e. '. m amount ofprotems) leads sometimes to alternate mterpretations, compansons between relative and absolute expressions showed similar trends. A Pharmacia SE-HPLC system (Pharmacia-Biotech S.A., 78280, Guyancourt, France) was used with a Superose-6 gelfiltration column to characterize the HPLC fractions from the two wheat varieties. Analysis was performed with the same buffer used for elution and protein extraction, with a 0.4-mL/ min ftow rate and 200-µL sample loading according to Lundh and MacRitchie (1989). Nine fractions containing protein material were collected every 2 min between 8 and 26 min of elution time and could be assigned to the regions Fl to F5 of the elution curve. Reproducibility of the method, previo~sly investigated by Dachkevitch and Autran (1989), was sufficient to observe small differences between samples. For instance, coefficients of variation of percentages were 1 to 2 % when loading the same protein extract several times and were 2 to 3 % when running different protein extracts of the same flour. Electrophoresis. Total proteins were extracted by a reducing solvent [1 M tris/HCl buffer (2-amino-2-hydroxymethyl1,3-propanediol), pH 6. 8, 20 g L -• SDS, 100 mL L -i glycerol, 0.1 g L - 1 pyronin (3,6-Bis(dimethylamino)xanthy~ium chloride), and 50 mL L- 1 2-mercaptoethanol] and frac~onated by 13% SDS-PAGE according to Autran and Bemer (1984). Subunit composition of SE-HPLC peaks was also determined by SOS-PAGE. To recover concentrated proteins from collected peaks, the SDS was removed by precipitating the protein fraction by 150 g L - •trichloroacetic acid according to Dachk~v­ itch (1989). The protein residue was washed two times with 1 mL of acetone and air dried. Dry protein extracts were dissolved in the Tris-SOS reducing buffer, then electrophoresed by 13 % SOS-PAGE. Percentag~ of the. main b~ds ~r groups of bands was estimated by densitometric scanning with a soft laser densitometer (Ultroscan 2002, LKB Instruments, Bromma Sweden) according to Autran et al. (1987). Because fast- and slow-moving fractions have different amino acid compositions and are likely to bind different proportions of Coomassie Blue, it is well known that the recorded band intensities may not reflect true amounts of the various protein fractions. However, intra-gel comparisons of homologous bands were shown to yield reliable and reproducible ( ± 2 %) results (Mecham et al., 1981; Autran et al., 1987). Nitrogen Determination. Nitrogen content of the ground seeds and extracts was determined by Kjeldahl analysis using a Cu-Se catalyst.

RESULTS AND DISCUSSION

Fraction N° 0

463

10

20

30

Elution time (min)

Fig. 1. Typical SE-HPLC elution pattern of durum wheat sto!a~e proteins extracted by phosphate-SDS buffer (pH 6.9). Arrows mdicate positions of molecular weight· references: Thyroglobulin (mol. wt. 669 kDa), alcohol dehydrogenase (mol. wt. 150 kDa), and bovine serum albumin (mol. wt. 66 kDa).

Changes in Electrophoretic Patterns. The SDSPAGE patterns of total reduced protein were determined for 15 sample~ of developing kernels for each cultivar, ranging from l: daily mean temperature (dmt) 200°C to maturity (Fig. 2). All main protein fractions of mature kernels were present at the earliest stages of kernel development. Patterns remained esse~tially s!8ble from the earliest stages of development until matunty, except

464

CROP SCIENCE, VOL. 34, MARCH-APRIL 1994

a. CAPDUR HMW -

94,000 64,000

ro-gliadins LMWtriplet

a,

43,000

p, y-g liadins 30,000

albumins

20,100 14,400

Ldmt°C

200 275 410 455 530 610 710 790 860 915

b. TOMCLAIR

R

--

HMW

MW

......

CJ>-gliadins LMW quadruplet

a,

p, y-gliad;ns

album;ns

94,000 6-1,000

[1

43,000

[

I

30,000

20, 100 14,400

Ldmt°C

200 275 410 455 530 610 7 10 790 860 915 R

MW

Fig. 2. SOS-PAGE patterns of reduced protein extracts from seeds of Capdur (a) and Tomclair (b) at different stages of development. ~t °C are indicated. R = mature seeds. MW = molecular weight reference mixture. Molecular weight markers are phosphorylase b, 94 000; bovine serum albumin, 68 000; ovalbumin, 43 000; carbonic anhydrase, 30 000; trypsin inhibitor, 20 100; a-lactalbumine, 14 400. Major polypeptides are indicated on the left.

for some variation in band intens1tJes. Such a lack of major changes in the electophoretic patterns is consistent with most results published concerning bread wheat (Feillet, 1965; Bushuk and Wrigley, 1971 ; Mecham et al. , 1981; Terce-Laforgue and Pernollet, 1982; Huebner et al., 1990) or durum wheat (Galterio et al., 1987). We attempted to further investigate quantitative variation of various protein fractions [HMW and LMW subunits of glutenin, main (a. + B+ y) gliadins, ro- gliadin ,

and albumins] through accurate densitometric scanning of the patterns (Table l ). Observed trends confirmed previous reports, namely (i) a rapid increase of gliadins until a maximum at Edmt 550 °C (i. e., about 4 wk post-anthesis), (ii) a later synthesis of glutenin subunits, and (iii) a decrease of albumins. Although most temperatures more than 30 °C were recorded after mid-June, during the dehydration step, some changes observed in relative amounts of gliadin and glutenin fractions may

·,

465

BENETRIX ET AL.: DURUM WHEAT PROTEIN COMPLEX CHANGES

Table 1. Changes in distribution of relative amount of main protein fractions [high (BMW) and low (LMW) molecular weight subunits of glutenin, (1)-gliadin, albumins, and (ci + P + y) gliadins] at four developmental stages ~t °C = 200, 410, 610, and mature seed) in endosperm samples from durum "Wheat cultivars Tomclair and Capdur. Total amounts do not reach 100% because only data on the most typical fractions are given. Total area of densitograms, % Developmental

stages

LMW

HMW glutenins

glutenins

0.6 1.2 1.9 1.6

12.2 16.4 17.9 24.4

0.3

22.3 22.0 25.5 26.5

:tdmt 200°c :tdmt 410°C :tdmt 610°C Mature seed :tdmt 200°c :tdmt 410°C :tdmt 610°C Mature seed

o.s 1.2 1.3

(a+ m-gliadin Capdur 1.8 1.9 2.7 1.7 Tomclair 2.2 2.4 3.5 2.0

have been the result of high temperatures (Blumenthal et al., 1990). However, no clear difference in the biosynthetic trend between the two cultivars was observed. Chromatographic Fractions. The SE-HPLC profiles of unreduced proteins extracted with sodium phosphateSDS buffer differed among the four developmental stages of each cultivar (Fig. 3). The magnitude of the excluded peak (Fl) progressively increased for both varieties as a.CAPDUR

p+

Albumins

gliadins

38.7 35.8 32.9 32.3

30.1 31.7 33.S 33.7

23.7 23.S 25.3 24.4

41.S 41.0 35.7 37.2

y)

the seeds developed, but this increase was steadier and more obvious for Capdur (Fig. 4). The percentage of Fraction F2 (intermediate aggregates) remained essentially unchanged in Tomclair ( = 15%), whereas in Capdur, F2 increased very rapidly from 11 to 21 % at about :Edmt 600°C, reaching a constant value ( =20%) during grain dehydration. Because large changes occurred in F2, this fraction was split into F2a (mol. wt. 250 000-600 000) and F2b (mol. wt. 100 000-250 000).

b. TOMCLAIR

(1)

a. Capdur

(1)

~

40

N

-...---

~

-+---a--

30

F1 F2 F2a

--o-- F2b 20 (2)

F4

-...---+--

Fl F2 F2a F2b F4

(2)

~

10

0

en

----A--

~

~en ~

0~~=11=~::::.~===-~ 100

'3c::

300

500

700

900

1100

I.dmt°C

G)

e

§'

(3)

~ '° ~

(3)

b. Tomclair

~ 0

40 30

~

--0--

(4)

(4)

----A--

20 10

o-l-~1;=::J1:1C=::!=:a==~~~_J 0

10

20

Elution time (min)

30

0

10

30

Elution time (min)

Fig. 3. Changes in SE-HPLC protein elution profiles (unreduced) from Capdur (a) and Tomclair (b) at three stages of development: (1) ldlmt °C 200, (2) = 410, and (3) = 610 and from mature seed (4).

=

100

300

500

700

900

1100

Ldmt°C

Fig. 4. Changes in percentages of some SE-HPLC fractions upon grain development and maturation from Capdur (a) and Tomclair (b), Fl (mol. wt. > 600 000); F2 (mol. wt. 100 000-600 000); Fla (mol. wt. 250 000-600 000); F2b (mol. wt. 100 000-250 000); F4 (mol. wt. 20 000-55 000).

466

CROP SCIENCE, VOL. 34, MARCH-APRIL 1994

50..,.-~~~~~~~~~~~~~

40

u:

30

-o-

~'i! 20

'-+----

Capdur Tomclair

10 o+--r-~.--~--.,.~~-,-~~--r-~---i

100

300

500

700

900

1100

Fig. S. Changes in estimated percentage of insoluble fraction (F1) in developing Capdur (0) and Tomclair (t) durum wheats.

For Capdur, before :Edmt 650 °C, proteins of molecular weight lower than 250 000 (F2b) were present in larger amounts than those higher molecular weight (F2a). During grain dehydration, an inversion of the slope occurred in chromatograms, indicating that Fraction F2a became predominant. In contrast, no inversion of slope was observed with Tomclair; F2a and F2b tended to be constant after :Edmt 700 °C . The main types ofmonomers (mo!. wt. 20 000-50 000) (Fraction F4) tended to increase in Tomclair during grain maturation, whereas in Capdur, this fraction decreased slightly in early stages of development and then remained essentially unchanged. In addition, because a portion of the highest molecular weight protein (glutenin) usually remains insoluble regardless of extraction conditions, changes in nitrogen Fi were followed by Kjeldahl analysis (Dachkevitch, 1989). The Fi increased in Capdur but remained essentially constant in Tomclair (Fig. 5) . During grain drying ,

Capdur, which produces a strong gluten, also had greater amounts of insoluble protein than did Tomclair, which produces a weaker gluten. This agrees with results for bread wheats obtained by Lundh and MacRitchie (1989). These results suggest that monomers or LMW aggregates were transformed into higher molecular weight complexes during kernel development. These changes were greater in Capdur than in Tomclair. Increased protein aggregation and insolubilization during development and dehydration appeared greater in high-quality than in low-quality cultivars. This conclusion is supported by opposite trends in (i) amount of Fi, (ii) percentage of excluded peak (Fl), (iii) percentage of monomeric fractions (F4), and (iv) inversion of slope in the profile of intermediate aggregates (F2). Protein Characterization. Compositions of protein in the nine fractions collected from the Superose-6 gelfiltration column were analyzed by SDS-PAGE with reduction by 2-mercaptoethanol in comparison with the whole phosphate-SOS extract and with the total reduced proteins (Fig. 6). The first SE-HPLC fraction (corresponding to the first region of the elution curve, i.e., excluded peak Fl) consisted essentially of HMW and LMW subunits of glutenin, whereas LMW glutenin subunits predominated in the next three fractions that made up the intermediate aggregates (F2). Small amounts of HMW glutenin subunits and gliadins were also present in F2. The two fractions of Peak F3 contained LMW glutenin subunits, w-gliadins, and a large amount of a-, !}-, and y-gliadins as well as some other fast-moving gliadins and albumins. Peak F4 contained only a, !}, and y types of gliadins with some fast-moving proteins,

94,000 64,000

43,000

30.000

20, 100 14 ,400

T

Fs

F1

F2

F3

F4

F5

MW

Fig. 6. SDS-PAGE patterns of reduced protein fractions of Tomclair collected from Superose-6 gel-filtration column. T, total reduced proteins; Fs, supernatant of phosphate-SOS extraction; Fl to FS, SE-HPLC peak number as in Figure l; MW, molecular weight standards as in Figure 2.

r

.

, 467

BENETRIX ET AL.: DURUM WHEAT PROTEIN COMPLEX CHANGES

and Peak F5 was made up of some fast-moving gliadins and albumins with still some a-, (}-, and y-gliadins eluting later. These compositions of the main SE-HPLC peaks corroborate previous results of Dachkevitch (1989) on bread wheats and especially the presence of LMW subunits in intermediate-sized aggregates F2 (100 000600 000). Our results emphasize the contribution of LMW subunits because Subfractions F2a and F2b (Fig. 4) were most clearly involved in changes in protein composition during grain development and dehydration. Likewise, LMW subunits were the ones most clearly showing differences between cultivars, indicating that these subunits may be major indicators of quality among durum wheats. CONCLUSIONS It appears that SE-HPLC is a powerful tool for studying native protein aggregates, providing information not available with electrophoresis techniques. With SEHPLC, we found that strong modifications occur mainly during grain dehydration, with increased level of aggregation. To our knowledge, this study is the first investigation of changes in protein complexes of durum wheat during seed development as they relate to seed quality. Our results provide a basis for further investigations of aggregate composition of wheat proteins in developing seed. In our results with two contrasting cultivars, several trends occurred: 1. Molecular size distributions of storage pr~teins changed throughout grain development, especially during grain dehydration. The trend was for increased size of protein aggregates. 2. There are noticeable differences in accumulation of large aggregates (insoluble residue and Fraction Fl), medium-sized aggregates (Fraction F2), and monomers (e.g., Fraction F4) between the two cultivars. For instance, increases in Fl, F2, and the insoluble residue were more apparent in Capdur than in Tomclair. 3. As suggested by inversion of the slope in the F2 region [i.e., the changes in the respective abundance of protein types F2a (mol. wt. 250 000-600 000) and F2b (mol. wt. 100000-250000) during development], the aggregation phenomenon largely involves LMW subunits of glutenin. The presence and proportion on the basis of total proteins of the LMW subunits of glutenin have been clearly associated with intrinsic pasta quality of durum wheat genotypes (Autran et al., 1987; Feillet et al., 1989). We postulate that the tendency of LMW subunits of glutenin to aggregate during grain dehydration may help explain differences found in pasta quality among durum wheat cultivars. Moreover, because it has been reported that the percentage of gliadins (Fraction F4) is greatly influenced by environment, it is likely that aggregation profiles, inferred from SE-HPLC, may reveal environmental effects within cultivars. Unlike SDS-PAGE, which primarily gives fingerprints of genotypes, SE-HPLC may help explain quality differences of cultivars grown under various climates and agricultural practices and may be of interest to many agronomists and chemists.

REFERENCES 'A.utran, J.C. 1994. Size-exclusion high-performance liquid chromatography for rapid examination of size differences of wheats and cereal proteins. Jn J.E. KrugerandJ.A. Bietz (ed.) HPLC of cereal and legumes proteins. Am. Assoc. Cereal Chem. St Paul, MN (In press). Autran, J.C., and R. Berrier. 1984. Durum wheat functional subunits revealed through heat treatments. Biochemical and genetic implications. p. 175-183. In A. GravelandandJ.H.E. Moonen (ed.) Proc. 2nd Intl. Workshop on Gluten Proteins, 1-3 May, Wageningen, the Netherlands. TNO, Wageningen. Autran, J.C., and G. Galterio. 1989. Associations between electrophoretic composition of proteins, quality characteristics and agronomic attributes of durum wheats. Il. Protein-quality associations. J. Cereal Sci. 9:195-215. Autran, J.C., B. Laignelet, and M.H. Morel. 1987. Characterization and quantitation oflow molecular weight glutenins in durum wheats. Biochimie 69:699-711. Blumenthal, C.S., I.L. Batey, F. Bekes, C.W. Wrigley, and E.W.R. Barlow. 1990. Gliadin genes contain heat-shock elements: Possible relevance to heat-induced changes in grain quality. J. Cereal Sci. 11:185-187. Bushuk, W., and C.W. Wrigley. 1971. Glutenin in developing wheat grain. Cereal Chem. 48:448-455. Cerning, J., and A. Guilbot. 1973. Changes in the carbohydrate composition during development and maturation of the wheat and barley kernel. Cereal Chem. 50:220-232. Dachkevitch, T. 1989. Etude des complexes proteiques de ble tendre par chromatographie liquide haute performance de tamisage moleculaire (SE-HPLC): Relation avec la qpalite technologique. These de Doctorat d'Etat, Universite des Sciences et Techniques du Languedoc, France. Dachkevitch, T., and J.C. Autran. 1989. Prediction of baking quality of bread wheats in breeding programs by size-exclusion highperformance liquid chromatography. Cereal Chem. 66:448-456. Damidaux, R., J.C. Autran, P. Grignac, and P. Feillet. 1978. Mise en evidence de relations applicables en selection entre l'electrophoregramme des gliadines et les proprietes viscoelastiques du gluten de Triticum durum Desf. C. R. Acad. Sci. Paris, Serie D, 287: 701-704. Feillet, P. 1965. Contribution l'etude des proteines du ble. Influence des facteurs genetiques, agronomiques et technologiques. These de Docteur-Ingenieur, Universite de Paris, Paris. Feillet, P. 1977. La qualite despites alimentaires. Ann. Nutr. Diet. 12:299-310. Feillet, P. 1988. Protein and enzyme composition of durum wheat. p. 93-119. In G. Fabriani and C. Lintas (ed.) Durum wheat: Chemistry and technology, Am. Assoc. Cereal Chem. St Paul, MN. Feillet, P., 0. Ait-Mouh, K. Kobrehel, and J.C. Autran. 1989. The role of low molecular weight glutenin proteins in the determination of cooking quality of pasta products: An overview. Cereal Chem. 66:26-30. Galterio, G., E. Biancolatte, and J.C. Autran. 1987. Proteins deposition in developing durum wheat. Implication in technological quality. Genet. Agr. 41:461-480. Huebner, F.R., J. Kaczkowski, and J.A. Bietz. 1990. Quantitative variation of wheat proteins from grain at different stages of maturity and from different spike locations. Cereal Chem. 67:464-470. Kaczkowski, J., H. Pior, J. Kwinta, S. Kos, and W. Bushuk. 1987. Some analytical data of protein behaviour in developing wheat kernel. p. 400-416. In R. Usztity and F. Bekes (ed.) Proc. 3rd Intl. Workshop on Gluten Proteins, 6-9 May, Budapest. World Scientific, Singapore. Lundh, G., and F. MacRitchie. 1989. Size-exclusion HPLC characterization of gluten protein fractions varying in breadmaking potential. J. Cereal Sci. 10:247-253. Mecham, D.K., J.G. Fullington, and F.C. Greene. 1981. Gliadin proteins in the developing wheat seed. J. Sci. Food Agric. 32: 773-780. Millin, B.J., J.M. Field, and P.R. Shewry. 1983. Cereal storage proteins and their effect on technological properties. p. 255-319. In J. Daussant et al. (ed.) Seed proteins. Phytochemical Society of Europe Symposia, Ser. 20, Chap. 12, Acad. Press, London.

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a

t'l'·· _..,

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Millet, M.O., A. Montembault, and J.C. Autran. 1991. Hordein composition differences in various anatomical regions of the kernel between two different barley types. Sci. Aliments 11: 155-161. Osborne, T.B. 1907. The proteins of wheat kernel. Carnegie Institute of Washington. Publ. 84. Pogna, N., D. Lafiandra, P. Feillet, and J.C. Autran. 1988. Evidence - for a direct causal effect of low molecular weight subunits of glutenins on gluten viscoelasticity in durum wheats. J. Cereal Sci. 7:211-214. Reeves, C.D., H.B. Krishnan, and T .W. Okita. 1986. Gene expression in developing wheat endosperm. Accumulation of gliadin and ADP glucose pyrophosphorylase messenger RNAs and polypeptides. Plant Physiol. 82:34-40.

Shewry, P.~.B.J. Millin, and D.D. Kasarda. 1984. The structural and evglutionary relationship of the prolamin storage proteins of barley, rye and wheat. Philos. Trans. R. Soc. London, B: 304: 297-308. Skerritt, J.H., P.Y. Lew, and S.L. Castle. 1988. Accumulation of gliadin and glutenin polypeptides during grain development of normal and sulfur-deficient wheat seed: Analysis using specific monoclonal antibodies. J. Exp. Bot. 39:723-737. Terce-Laforgue, T., and J.C. Pernollet. 1982. Etude quaptitative et qualitative de !'accumulation des gliadines au cours du developpement du caryopse de ble (Triticum aestivum L.). C. R. Acad. Sci. Paris, 294, Serie ill, 529-534.