Gálová, Z., Michalik, L, and Å molková, H.: Biosyn- téza vysoko a nÃzkomolekulárnych gluténových sub- jednotiek ako molekulárnych markerov chleb op ekárskej.
Effect of nitrogen fertilization on the content of H M W glutenin subunits in winter wheat grain (Triticum aestivum L.) Z. GÁLOVÁ, H. ŠMOLKOVÁ, and I. MICHALÍK
Department
of Biochemistry
and Biotechnology,
Faculty of Agronomy, Slovak Agricultural
Studying the effect of nitrogen fertilization on the content of proteins in common winter wheat grain, the highest proteins content increase has been established by full fertilization variant (average 10%). The influence of nitrogen fertilization is cultivar-dependent. The good bread-making quality cultivars utilize nitrogen less intensively as cultivars with feed quality. INTRODUCTION All the important phenomena that occur during the grain filling depend not only on the maturation conditions (temperature and water disposability), which were studied by authors as Marchylo et al [5] and Gálová et al [3], but also on nitrogen fertilization. Nitrogen nutrition is largely considered as the main factor affecting storage proteins as well as the technological quality of wheat samples. According to prevent studies [6, 10], the amount of storage proteins affects both the sources of nitrogen fertilization and the application techniques. Doekes et al [2] and Levi et al [4] observed an increase in gliadins but no change of glutenins amount upon nitrogen fertilization. Scheromn et al [12] revealed a varietal-dependent increase of both glutenins and gliadins. A varietaldependent effect of nitrogen fertilization was also observed by Prieto et al [11]. The aim of the present work was to evaluate the effect of four nitrogen fertilization variants on two common winter wheat cultivars Regia and Sana 1000 grains weight, protein content and electrophoretically determined HMW glutenin subunits, individual (pairs) HMW glutenin subunits and other subunits content in 1997 year.
University, SK-949 16 Nitra
b) rational of fertilization variant - the application of regenerative N ration (2/5 of the total N rate calculated, in the form of ammonium nitrate, before the tillering time) and productive N ration (3/5 one at the shooting time), total N rate was calculated according to the 5 t / h a harvest norms and was applicated with regard to the N inorganic soil content c) diagnostic of the fertilization variant [7] d) fertilization variant - the application of sowing ploughing nitrogen ration (1/5 of the total N rate calculated, in the form of ammonium sulphate), regenerative N ration (2/5 in the form of ammonium nitrate, before the tillering time) and productive N ration (2/5 in the form of the liquid organic mineral leaf fertilizer Unihum-N, at the shooting time), total N rate was calculated according to the 5 t/ha harvest norms. Full physiological mature samples were dehulled and hull-free grains were milled in a laboratory mill (Mill 14 920, A/S N. Foss Electric, Denmark). Flour samples were analyzed by standard A AC С proce dures [1] for crude protein (method 46-13). The spe cific nitrogen-to-protein conversion factor was 5,7. Glutenin subunits of high molecular weight were frac tionated from the grains by standard vertical discontinual electrophoresis (gel electrophoresis appa ratus SE 660, Pharmacia Biotech) in the Polyacry lamide gel in the presence of SDS reported by in ternational organisation ISTA, ISTA SDS-PAGE [13] with the next densitometric detection (ImageMaster DTS, Pharmacia-LKB, programme Diversity One, PDI). For the identification of the HMW glutenin subunits the nomenclature proposed by Payne et al [8] was used.
MATERIALS AND METHODS RESULTS A N D DISCUSSION The accessions evaluated in this study were two common winter wheat cultivars Regia and Sana (Triticum aestivum L.). The wheats were grown in a five-replicate randomized (12 m 2 ) complete block experiment at experimental field Dolná Malanta in southern Slovakia in 1996/97 growing season with feed pea forecrop and Aminex and Superbarnon herbicide treatment. Nitrogen nutrition was applied according to the following parameters: a) no fertilization variant
Chem. Papers 52 (Focus Issue) 585—586 (1998)
It is well established that the nitrogen fertilization level influences the accumulation of proteins in wheat grains. The increased protein quantity leads to im proved quality parameters (especially dough develop ment and bread volume). As the results from the ta ble 1 show the positive effect of nitrogen fertilization was determined by every cultivar and by all variants of nitrogen treatment. The highest proteins content increase has been established by full fertilization vari-
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Table 1. 1000 grains weight, proteins flour content and HMW glutenin subunits, individual (pairs) glutenin subunits and other electrophoreticaly divided subunits grain content of two common winter wheat (Triticum aestivum L.) cultivars Regia and Sana in 1997 year. Cultivar Nitrogen level
A
1000 grain weight [g] e Proteins [%] f HMW glu. sub. [%] f subunit 1 [%] f subunits 7+9 [%] f subunits 2+12 [%] f subunits 5+10 [%] f other subunits^ [%] a
a
Sana (Glu -score 5) b c B C
48,41 8,00 7,1 1Д 2,2
47,12 8,40 8,7 1,3 3,0
3,8 70,5
4,4 67,6
ns
48,32 8,40 6,8 1,0 2,4
ns
3,4 58,9
D
d
49,60 8,72 5,3 0,9 2,3 2,1 69,0
A ns
a
Regia (Glu-score 9) b c B C ns
D
51,79 9,19 4,5
50,08* 9,59 5,3
50,34 9,91 5,0
2,1 2,4
2,3 3,0
2,2 2,8
2,4 3,1
69,9
66,1
68,2
68,8
b
d
49,38** 10,24 5,5
d
no fertilization variant, rational of fertilization variant (Materials and Methods), diagnostic fertilization variant [7], full fertil e f g ization variant (Materials and Methods), % of dry basis, relative % (% of all electrophoreticaly divided subunits), LMW subunits ns + gliadins + Alb+Glo, not significant, *'** significant at P < 0.05, P < 0.01, respectively
ant D (9,0% and 11,4%, Regia and Sana, resp.), which was combined with the significant 1000 grain weight decrease by cultivar Sana. All values for proteins are low because of rainy climatic conditions in the time of maturation and harvest in 1997. As regards the influence of nitrogen fertilization to storage proteins content we can see the cultivar-dependent differences previously reported by Scheromn et al [12]. There is HMW glutenin subunits increase by the cultivar Regia in rational fertilization variant В and HMW glutenin subunits decrease in the diagnostic fertilization vari ant С and the full fertilization variant D unlike the cul tivar Sana in all fertilization variants HMW glutenin subunits increase. The same situation was determined by the individual HMW glutenin subunits composi tion. Other subunits content decreases in all fertiliza tion variants. Generally we can say, that the common winter wheat cultivars (Regia) of good bread-making quality do not utilize nitrogen so intensively as culti vars (Sana) of weak quality. It is still unclear which of the gluten protein components determines qual ity. Pechanek et al [9] proposed that the best early detectable parameter with the best predictive value for bread-making quality could be the ratio of HMW glutenin subunits, especially the x-type subunits, to the total protein content because of the strong corre lation between these parameters. Acknowledgements. This work was supported by grant VTP 95/5Ц5/683.
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REFERENCES 1. American Association of Cereal Chemists. The Associ ation, St. Paul, MN, 1995. 2. Doekes, G. J., and Wennekes, L. M. J. Cereal Chem. 59, 276 (1982). 3. Gálová, Z., Michalik, L, and Šmolková, H.: Biosyntéza vysoko a nízkomolekulárnych gluténových subjednotiek ako molekulárnych markerov chleb op ekárskej kvality zrna pšenice. [Záverečná správa]. Nitra, SPU 1996. 32 s. 4. Levi, A. A., Galili, G., and Feldman, M., Theor. Appl. Genet. 69, 429 (1985). 5. Marchylo, B. A., Kruger, J. E., and Hatcher, D. W. Cereal Chem. 67, 372 (1990). 6. Martin, G., and Taureau, J. C , Perspectives Agricoles 165, 16 (1992). 7. Michalik, L, and Ložek, O., Rostlinná výroba 31, 487 (1985). 8. Payne, P. L, and Lawrence, G. J. Cereal Research Communications 11, 29 (1983). 9. Pechanek, U. Karger, A., Groger, S., Charvát, В., Schoggl, G., and Lelley, Т., Cereal Chem. 74, 800 (1997). 10. Peltonen, J., Acta Agric. Scand. Sect. B, Soil and Plant Sei. 45, 2 (1995). 11. Prieto, J. A., Kelfkens, M., Weegels, P. L., and Hamer, R., Z. Lebensm. Unters. Forsch. 194, 337 (1992). 12. Scheromm, P., Martin, G., Bergoin, A., and Autran, J.-C, Cereal Chem. 69, 664 (1992). 13. Wrigley, C. W., in: Seed analysis, pp. 17-41. Springer - Verlag, Berlin, Heilderberg, 1992.
Chem. Papers 52 (Focus Issue) 585—586 (1998)
