Malting quality studied by I E F and I P G - D a l ~
E l r i l r o p h u i c o s 1992, 13, 759-770
Angelika Gorg Wilhelm Postel Walter Weiss Technische Universitat Munchen, Lehrstuhl fur Allgemeine Lebensmitteltechnologie, Freising-Weihenstephan
Detection of polypeptides and amylase isoenzyme modifications related to malting quality during malting process of barley by two-dimensional electrophoresis and isoelectric focusing with immobilized pH gradients Two cultivars (“Alexis” and “Lenka”) of contrasting final attenuation values were malted, and the protein and amylase isoenzyme composition, as well as the change in protein and amylase isoenzyme composition during malting, was investigated by two-dimensional polyacrylamide gel electrophoresis of total proteins, and isoelectric focusing of amylase isoenzymes, respectively. Isoelectric focusing demonstrated that significant differences exist between the amylase isoenzyme patterns of the two cultivars, suggesting a correlation between the presence of certain amylase isoenzyme bands and final attenuation. This finding was confirmed by analysis of 36 barley cultivars with a wide range of quality. It was shown that all cultivars which are of low or, at best, moderate final attenuation values exhibit the amylase band “B” (isoelectric point = 6.8), whereas those cultivars which are predominantly of high malting grade do not possess this “B”isoenzyme band, but exhibit the pronounced “A” isoenzyme band (isoelectric point = 6.5) instead, suggesting that these isoenzymes (which we suppose to be 6-amylases) can be utilized to predict the final attenuation values of unknown barley samples or new lines. However, “final attenuation” is a complex function. Preliminary results of two-dimensional gel electrophoresis indicate that other factors, such as total amount of amylases, or a 19 kDa A hordein-like polypeptide, which was degraded faster in the low malting grade cultivar “Lenka”, may also have a role in determining quality.
1 Introduction The bread- and pasta-making quality of wheat (Triticumsp.) cultivars is a function of their protein composition. It has been shown that the presence of particular wheat storage proteins correlates with baking or pasta-making quality, and this finding has enabled the application of protein electrophoretic techniques for selection purposes in wheat breeding (see [ 11for a review). Similarly, a major goal in barley (Hordeurn vulgare L.) research is to identify proteins which are related to malting and brewing quality. With the help of suitable genetic markers, quality selection would be much easier for barley breeders, at an early stage of a plant breeding program, without having to apply time-consuming field trials and technological tests, which require relatively large amounts of seed material. By analogy to wheat, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and acid-PAGE have been used to reveal a possible relationship between the storage proteins of barley (the hordeins) and malting quality [2-41. However, in contrast to the successful application of protein electrophoretic techniques for the estimation of quality in wheat, ultimately almost all efforts to con-
Correspondence: Priv. Doz. Dr. A. Gorg, Technische Universitiit Munchen, Lehrstuhl fur Allgemeine Lebensmitteltechnologie, DW-8050 Freising-Weihenstephan, Germany Abbreviations: cv, cultivar; DTT, dithiothreitol; IEF, isoelectric focusing; IPG, immobilized pH gradient; IPG-DALT, two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradient in the first dimension; M,, relative molecular mass: PAGE, polyacrylamide gel electrophoresis; PI,isoelectric point; S D S , sodium dodecyl sulfate; Tris,Tris(hydroxymethy1)aminomethane; 2-D, two-dimensional
0VCH Verlagsgesellschaft
759
mbH, D-6940 Weinheim, 1992
firm a strong relationship between barley storage protein patterns and malting quality failed. The only exception from this general rule is the finding that the amount of a small hordein fraction (primarily D hordeins) in malt, and the rapidness of degradation of these proteins during malting, seems to be associated with malting quality in terms of malt extract yield [5]. Maltig quality is a complex function. Besides total protein content (which is also strongly influenced by environmental factors) and malt extract yield, final attenuation is one of the most important quality criteria for malting barleys in Germany. For this reason, we examined the relationships between particular barley seed proteins, as well as the rate and extent of seed protein modification during germination and malting, and the malting quality character final attenuation [6-71. However, in these studies we were unable to demonstrate a close correlation between final attenuation values and the rate of hordein protein breakdown in the cultivars examined. In these experiments, in which SDS-PAGE of different solubility fractions of barley seed and malt proteins was utilized,we found that the concentration of aqueous-alcohol soluble storage proteins, the hordeins, diminished considerably during the malting process (independently from the malting quality, however), whereas the albumins and globulins were much less affected, with the exception of two new polypeptide bands which appeared during malting. We suppose that these polypeptides ( M r = 43 kDa and M, = 60 kDa) are a- and 6-amylases which are synthesized de nova or released from higher M, precursors, respectively. A large number of a-amylase isoenzymes, which exhibit identical relative molecular masses, but different isoelectric points, has been described [S-1 I], and the same is true 0173-0835/Y2/0Y 10-0759 $3.50+.25/0
for P-amylases [12-141. The best isoenzymes (or polymorphic proteins) to use as genetic markers are those which exhibit multiple bands upon electrophoresis. Therefore, more powerfully resolving techniques than SDSPAGE are likely to reveal more polymorphism at enzyme loci [l]. SDS-PAGE resolves proteins according to size ( M J , and is thererore not an appropriate electrophoretic separation method for these isoenzymes. In the present study, two-dimensional (2-D) PAGE with immobilized pH gradients (IPG-DALT) and isoelectric focusing (IEF) were used or the separation of barley seed and barley malt proteins, and amylase isoenzymes, respectively. The proteins and enzymes had been extracted from samples of two barley cultivars of contrasting final attenuation values, which had been withdrawn at daily intervals during a micro-scale malting experiment of these cultivars. Should a correlation be found between the presence of particular barley seed or malt proteins, or amylase isoenzymes as assessed by IPGDALT or IEF, and the malting quality character final attenuation, these preliminary investigations could be followed by a survey of a larger number of barley cultivars of known quality. The spring barley cultivars used in this survey were also similar in most of the more important quality characters (such as total protein content or malt extract yield), but differed in their final attenuation values, so that the differences between their final attenuation values were supposed to be due exclusively to the differences in their protein or enzyme composition.
2 Materials and methods 2.1 Apparatus and chemicals
Equipment for IEF and IPG-DALT (Multiphor I1 electrophoresis chamber, Macrodrive V power supply, Multitemp I1 thermostatic circulator, gradient mixer), ImmobilinesR, 2-D Pharinalytes pH 3-10, p l marker proteins, Ultrodex and GelBond PAGfilm were from Pharmacia-LKB (Uppsala, Sweden). Electrode wicks (ultra wicks) for horizontal SDS-PAGE were from Bio-Rad (Richmond, CA, USA). Acrylamide (2 X cryst.), N,N-methylenebisacrylamide, ammonium persulfate, N,N,N”’-tetramethylethylenedianiine (TEMED), glycine, SDS and Triton X-100 were from Serva (Heidelberg, Germany). Tris(hydroxymethy1)aminomethane (Tris), dithiothreitol (DTT) and iodoacetamide were from Sigma (St. Louis, MO, USA). Urea, glycerol, sorbitol, soluble starch, iodine solution, silver nitrate and all other chemicals (analytical grade) for electrophoresis and staining purposes were from Merck (Darmstadt, Germany). 2.2 Seed material
Samples of 17 European spring barley cultivars, comprising a wide range of different final attenuation values, were provided by Dr. M. Baumer (Bayerische Landesanstalt fur Bodenkultur und Pflanzenbau, Freising-Weihenstephan, Germany). These cultivars are listed in Table 1, together with their potential malting grades. For additional analyses, 7 two-rowed winter barleys of low, and 12 six-rowed winter barley cultivars of very low final attenuation were also tested. These cultivars are listed in Tables 2 and 3, respectively.
Table 1. List o f two-rowed spring barley cultivars used in this study“’ Cu I t i v a r
Malt extract
Final attenuation
8-Amylase isoenzyrne
Alexis Arena Aura Fink Ursel Ultra Dorctt R Uni ba Beate Steina Ardni i r Defra Phantom Perun Lenka Lerche Berolina
9 9 8 8 8 8 9
8 7 1 1 7 6 6 6 6 5 5 5 5
“A
9 8 7 6 8 9
4 4 4 4
9 9 8 7
“A“ “A” “‘A”
“A “A“ “A” “A” “A“ “A”
“A“ “B” “B” “B” “B” “B” “B”
a) All malting grade data were taken from [37];scale: 1-9; 9 = b e s t ; arnylase isoenzymes “A”and “R”, see Fig. 7
Table 2. List of two-rowed winter barley cultivars used in this s t u d y ’ Cultivar
Malt extract
Kaskade Igri Marylin Diana Sonja Sonate Viola
6 3 3 2 3 1 1
Final attenuation
P-Amylase isoenzyme ___
“B” “B”
“B” “B” “B” “B”
“B”
a j All maltinggrade data were taken from [37]: scale: 1-9 (9 =best); amylase isoenzyme “B”, see Fig. 7
Table 3. List of six-rowed winter barley cultivars used in this study Banjo, Birgit, Brunhild, Dura, Franka, Gerbel, Hasso, Mammut, Ogra, Tapir, Triton, Vogelsanger Gold No exact malting grade data are available for these cullivars. but their malt extract and final attenuation values are very low throughout (values
E l r i l r o p h u i c o s 1992, 13, 759-770
Angelika Gorg Wilhelm Postel Walter Weiss Technische Universitat Munchen, Lehrstuhl fur Allgemeine Lebensmitteltechnologie, Freising-Weihenstephan
Detection of polypeptides and amylase isoenzyme modifications related to malting quality during malting process of barley by two-dimensional electrophoresis and isoelectric focusing with immobilized pH gradients Two cultivars (“Alexis” and “Lenka”) of contrasting final attenuation values were malted, and the protein and amylase isoenzyme composition, as well as the change in protein and amylase isoenzyme composition during malting, was investigated by two-dimensional polyacrylamide gel electrophoresis of total proteins, and isoelectric focusing of amylase isoenzymes, respectively. Isoelectric focusing demonstrated that significant differences exist between the amylase isoenzyme patterns of the two cultivars, suggesting a correlation between the presence of certain amylase isoenzyme bands and final attenuation. This finding was confirmed by analysis of 36 barley cultivars with a wide range of quality. It was shown that all cultivars which are of low or, at best, moderate final attenuation values exhibit the amylase band “B” (isoelectric point = 6.8), whereas those cultivars which are predominantly of high malting grade do not possess this “B”isoenzyme band, but exhibit the pronounced “A” isoenzyme band (isoelectric point = 6.5) instead, suggesting that these isoenzymes (which we suppose to be 6-amylases) can be utilized to predict the final attenuation values of unknown barley samples or new lines. However, “final attenuation” is a complex function. Preliminary results of two-dimensional gel electrophoresis indicate that other factors, such as total amount of amylases, or a 19 kDa A hordein-like polypeptide, which was degraded faster in the low malting grade cultivar “Lenka”, may also have a role in determining quality.
1 Introduction The bread- and pasta-making quality of wheat (Triticumsp.) cultivars is a function of their protein composition. It has been shown that the presence of particular wheat storage proteins correlates with baking or pasta-making quality, and this finding has enabled the application of protein electrophoretic techniques for selection purposes in wheat breeding (see [ 11for a review). Similarly, a major goal in barley (Hordeurn vulgare L.) research is to identify proteins which are related to malting and brewing quality. With the help of suitable genetic markers, quality selection would be much easier for barley breeders, at an early stage of a plant breeding program, without having to apply time-consuming field trials and technological tests, which require relatively large amounts of seed material. By analogy to wheat, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and acid-PAGE have been used to reveal a possible relationship between the storage proteins of barley (the hordeins) and malting quality [2-41. However, in contrast to the successful application of protein electrophoretic techniques for the estimation of quality in wheat, ultimately almost all efforts to con-
Correspondence: Priv. Doz. Dr. A. Gorg, Technische Universitiit Munchen, Lehrstuhl fur Allgemeine Lebensmitteltechnologie, DW-8050 Freising-Weihenstephan, Germany Abbreviations: cv, cultivar; DTT, dithiothreitol; IEF, isoelectric focusing; IPG, immobilized pH gradient; IPG-DALT, two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradient in the first dimension; M,, relative molecular mass: PAGE, polyacrylamide gel electrophoresis; PI,isoelectric point; S D S , sodium dodecyl sulfate; Tris,Tris(hydroxymethy1)aminomethane; 2-D, two-dimensional
0VCH Verlagsgesellschaft
759
mbH, D-6940 Weinheim, 1992
firm a strong relationship between barley storage protein patterns and malting quality failed. The only exception from this general rule is the finding that the amount of a small hordein fraction (primarily D hordeins) in malt, and the rapidness of degradation of these proteins during malting, seems to be associated with malting quality in terms of malt extract yield [5]. Maltig quality is a complex function. Besides total protein content (which is also strongly influenced by environmental factors) and malt extract yield, final attenuation is one of the most important quality criteria for malting barleys in Germany. For this reason, we examined the relationships between particular barley seed proteins, as well as the rate and extent of seed protein modification during germination and malting, and the malting quality character final attenuation [6-71. However, in these studies we were unable to demonstrate a close correlation between final attenuation values and the rate of hordein protein breakdown in the cultivars examined. In these experiments, in which SDS-PAGE of different solubility fractions of barley seed and malt proteins was utilized,we found that the concentration of aqueous-alcohol soluble storage proteins, the hordeins, diminished considerably during the malting process (independently from the malting quality, however), whereas the albumins and globulins were much less affected, with the exception of two new polypeptide bands which appeared during malting. We suppose that these polypeptides ( M r = 43 kDa and M, = 60 kDa) are a- and 6-amylases which are synthesized de nova or released from higher M, precursors, respectively. A large number of a-amylase isoenzymes, which exhibit identical relative molecular masses, but different isoelectric points, has been described [S-1 I], and the same is true 0173-0835/Y2/0Y 10-0759 $3.50+.25/0
for P-amylases [12-141. The best isoenzymes (or polymorphic proteins) to use as genetic markers are those which exhibit multiple bands upon electrophoresis. Therefore, more powerfully resolving techniques than SDSPAGE are likely to reveal more polymorphism at enzyme loci [l]. SDS-PAGE resolves proteins according to size ( M J , and is thererore not an appropriate electrophoretic separation method for these isoenzymes. In the present study, two-dimensional (2-D) PAGE with immobilized pH gradients (IPG-DALT) and isoelectric focusing (IEF) were used or the separation of barley seed and barley malt proteins, and amylase isoenzymes, respectively. The proteins and enzymes had been extracted from samples of two barley cultivars of contrasting final attenuation values, which had been withdrawn at daily intervals during a micro-scale malting experiment of these cultivars. Should a correlation be found between the presence of particular barley seed or malt proteins, or amylase isoenzymes as assessed by IPGDALT or IEF, and the malting quality character final attenuation, these preliminary investigations could be followed by a survey of a larger number of barley cultivars of known quality. The spring barley cultivars used in this survey were also similar in most of the more important quality characters (such as total protein content or malt extract yield), but differed in their final attenuation values, so that the differences between their final attenuation values were supposed to be due exclusively to the differences in their protein or enzyme composition.
2 Materials and methods 2.1 Apparatus and chemicals
Equipment for IEF and IPG-DALT (Multiphor I1 electrophoresis chamber, Macrodrive V power supply, Multitemp I1 thermostatic circulator, gradient mixer), ImmobilinesR, 2-D Pharinalytes pH 3-10, p l marker proteins, Ultrodex and GelBond PAGfilm were from Pharmacia-LKB (Uppsala, Sweden). Electrode wicks (ultra wicks) for horizontal SDS-PAGE were from Bio-Rad (Richmond, CA, USA). Acrylamide (2 X cryst.), N,N-methylenebisacrylamide, ammonium persulfate, N,N,N”’-tetramethylethylenedianiine (TEMED), glycine, SDS and Triton X-100 were from Serva (Heidelberg, Germany). Tris(hydroxymethy1)aminomethane (Tris), dithiothreitol (DTT) and iodoacetamide were from Sigma (St. Louis, MO, USA). Urea, glycerol, sorbitol, soluble starch, iodine solution, silver nitrate and all other chemicals (analytical grade) for electrophoresis and staining purposes were from Merck (Darmstadt, Germany). 2.2 Seed material
Samples of 17 European spring barley cultivars, comprising a wide range of different final attenuation values, were provided by Dr. M. Baumer (Bayerische Landesanstalt fur Bodenkultur und Pflanzenbau, Freising-Weihenstephan, Germany). These cultivars are listed in Table 1, together with their potential malting grades. For additional analyses, 7 two-rowed winter barleys of low, and 12 six-rowed winter barley cultivars of very low final attenuation were also tested. These cultivars are listed in Tables 2 and 3, respectively.
Table 1. List o f two-rowed spring barley cultivars used in this study“’ Cu I t i v a r
Malt extract
Final attenuation
8-Amylase isoenzyrne
Alexis Arena Aura Fink Ursel Ultra Dorctt R Uni ba Beate Steina Ardni i r Defra Phantom Perun Lenka Lerche Berolina
9 9 8 8 8 8 9
8 7 1 1 7 6 6 6 6 5 5 5 5
“A
9 8 7 6 8 9
4 4 4 4
9 9 8 7
“A“ “A” “‘A”
“A “A“ “A” “A” “A“ “A”
“A“ “B” “B” “B” “B” “B” “B”
a) All malting grade data were taken from [37];scale: 1-9; 9 = b e s t ; arnylase isoenzymes “A”and “R”, see Fig. 7
Table 2. List of two-rowed winter barley cultivars used in this s t u d y ’ Cultivar
Malt extract
Kaskade Igri Marylin Diana Sonja Sonate Viola
6 3 3 2 3 1 1
Final attenuation
P-Amylase isoenzyme ___
“B” “B”
“B” “B” “B” “B”
“B”
a j All maltinggrade data were taken from [37]: scale: 1-9 (9 =best); amylase isoenzyme “B”, see Fig. 7
Table 3. List of six-rowed winter barley cultivars used in this study Banjo, Birgit, Brunhild, Dura, Franka, Gerbel, Hasso, Mammut, Ogra, Tapir, Triton, Vogelsanger Gold No exact malting grade data are available for these cullivars. but their malt extract and final attenuation values are very low throughout (values
