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HOW IS GRAIN PROTEIN FORMED?

R.A. Spurway

Agricultural Research Institute, Wagga Wagga

Introduction

Proteins are complex organic nitrogenous substances which are present in the cells of all plants and animals and participate in all important physiological processes. Some examples of the great diversity in protein structure and function are:

• proteins of the gluten complex in dough which, through their elastic and flow properties, are of unique value in baking;

• fibrous proteins such as hair, wool and collagen, which are important structural elements of animal tissues;

• enzymes, antibodies and hormones are globular proteins which have important physiological functions in plant and animal tissues;

• conjugated proteins, such as those combined with nucleic acids and chlorophyll, have a central role in gene replication and photosynthesis.

Despite this diversity, all proteins share some basic features. They are high molecular weight molecules which invariably contain carbon, hydrogen, oxygen and nitrogen. Some, though not all, also contain sulphur and a few also contain phosphorus. The basic elements are in the form of amine and carboxyl groups which are linked to carbon atoms to form amino acids. Amino acids are the fundamental units of protein.

Morphology And Chemical Composition Of The Wheat Grain

A mature cereal grain is the reservoir of nutrients and products of biosynthesis which have been accumulated by the plant during its lifetime. During vegetative growth the continuous flow of water and nutrients through the roots and stems, together with the carbon taken up from the atmosphere, provides the raw products for the synthesis of proteins and carbohydrates. As each leaf begins to senesce, the proteins and carbohydrates are remobilised to provide the raw materials for new plant tissue. When vegetative growth of the plant gives way to grain filling (reproductive growth), the nitrogen required for grain protein synthesis is derived from senescing leaves, and in particular the flag leaf, by hydrolysis and transport as amino acids. Complex carbohydrates in stems and leaves are hydrolysed to simple sugars and transported to the grain for accumulation as starch.

The chemical composition of mature wheat grain is dominated by a high starch content, typically about 72% of the total dry weight (e.g. Simmonds, 1978; Lasztity, 1984) and a protein content between 6-16% (Simmonds, 1981). Starch is present only in the endosperm, but protein is distributed through all parts of the grain (Figure 1) as shown in Table 1.

Modern milling techniques enable most of the pericarp, testa and aleurone layer to be extracted as bran, the embryo and scutellum as wheat germ and the endosperm as flour.

Figure 1. A longitudinal section through the crease of the wheat kernel showing the major structures.

Table 1. Distribution of protein and starch in the wheat kernel (based on Hinton (1953) and MacMasters et al. (1971).

Fraction of kernel

% of kernel weight

(by dissection)

% of total starch

% of total protein

Pericarp, testa

8

0

4.5

Aleurone

7

0

15.5

Endosperm

82.5

100

72.0

Scutellum

1.5

0

4.5

Embryo

1

0

3.5

Proteins differ in their physical and biological properties in different fractions of the kernel. Comprehensive studies by Osborne more than 80 years ago (Osborne, 1907), showed that proteins in wheat differed in their solubility:

• albumins are soluble in water;

• glubulins are soluble in salt solutions but insoluble in water;

• gliadins are soluble in 70-90% ethanol;

• glutenins are insoluble in neutral aqueous solutions, saline solutions or alcohol.

In terms of their biological function, the albumins and globulins are largely the cytoplasmic or metabolically active proteins and the gliadins and glutenins are largely storage proteins (Lasztity, 1984). Storage proteins are typically endosperm or wheat flour proteins and the metabolically active proteins are found in the germ and pericarp -aleurone layers.

There are large differences in the amino acid composition of cytoplasmic and storage proteins (Table 2). The storage proteins contain a large proportion of glutamic acid and proline and only a small proportion of lysine, arginine, threonine and tryptophan. Metabolically active proteins contain considerably less glutamic acid and proline and have higher proportions of lysine and arginine which give these proteins a higher nutritive value, but lower functional (bre&lmaking) properties.

Table 2. Amino acid composition of wheat grain, flour, germ and aleurone proteins (g/100 g protein)

Amino acid

Wheat grain a

Flourb

Whole germc

Aleuroned

Alanine

3.6

2.6

7.00

6.5

Arginine

4.6

3.1

8.96

12.3

Aspartic acid

4.9

3.7

10.21

9.3

Cystine

2.5

2.8

0.66

-

Glutamic acid

29.9

34.7

15.45

18.3

Glycine

4.1

3.4

6.54

7.0

Histidine

2.3

1.9

2.63

4.3

Isoleucine

3.3

3.1

3.91

3.2

Leucine

6.7

6.6

6.79

6.7

Lysine

2.9

1.9

7.76

5.9

Methionine

1.5

1.3

1.88

0.4

Phenylalanine

4.5

4.8

4.07

4.2

Proline

9.9

11.8

4.37

4.6

Serine

4.6

4.4

4.62

5.0

Threonine

2.9

2.4

4.82

3.8

Tryptophan

1.1

1.5

-

-

Tyrosine

3.0

2.8

3.12

2.8

Valine

4.4

3.4

5.65

5.5

a From FAQ (1970)
b From Bushuk and Wrigley (1974)
c From Pomeranz et al. (197) (g/100 g amino acids)
d From Fulcher et al (1972)

Grain Development And Protein Synthesis

The metabolically active proteins, mainly albumins and globulins, are synthesised rapidly in the early stages of kernel development. This synthesis is associated with the early development of the embryo and aleurone layer, the latter being clearly differentiated from the outer layer of endosperm cells at 12-14 days after anthesis (Simmonds, 1978). In total, these proteins usually comprise less than 20% of the mature kernel (Konzak, 1977).

Five or six layers of large, vacuolated cells appear in the endosperm during the 4-5 days after anthesis and, at the same stage, starch accumulation will have commenced in the central cells (Simmonds, 1978). Storage protein first appears in the developing endosperm about 10 days after anthesis (Buttrose, 1963) and is synthesised continuously until the kernel has reached maturity (Jennings and Morton, 1963; Mitra and Bhatia, 1973). The rate of protein synthesis is initially high so that approximately half of the total storage protein is synthesised in the 20 days after anthesis (Simmonds, 1978). The last endosperm cells to be differentiated, the subaleurone cells, often have high protein contents and only small amounts of starch (Kent, 1966) whereas the earliest cells formed have high starch contents.

The protein bodies in developing endosperm cells are spherical, ranging up to 20 μm in diameter in mature tissue. However, their mature shape is extensively distorted due to dehydration of the kernel and compression by the harder starch granules, so that they tend to form a continuous fused matrix within which the starch granules are embedded (Lasztity, 1984). The association between starch granules and the protein matrix has a significant effect on the vitreous properties of grain and on kernel hardness.

Vitreousness is an optical phenomenon which is closely associated with a high protein content in the sub-aleurone endosperm. When the starch and protein adhere closely to each other, as in hard grains, light rays are not scattered at the surface of the endosperm and the grain appears vitreous (Duvick, 1961). On the other hand, the more open texture of soft grains is opaque. Vitreous and opaque areas may occur in different parts of the same endosperm, resulting in the often seen, but undesirable, mottling effect.

Grain hardness is genetically determined (Symes, 1969), suggesting that apart from the influence of physical factors on the texture of the endosperm, some biochemical factors also play an important role, at least in some species (Simmonds, 1978). Barlow et al. (1973) and Simmonds et al. (1973) concluded that the hardness of wheat kernels was partly due to a higher degree of adhesion between starch granules and the surrounding protein matrix in hard wheats as compared with soft wheats. They also observed residual proteins associated with the surfaces of starch granules in the endosperm which led to the suggestion that they could contribute to the strength of the starch/protein interfacial bond.

Practical Implications For Crop N Requirements

In developing cereal grains, early development of the embryo structures is followed by growth of the endosperm. This pattern ensures that if environmental conditions, such as drought or early death of leaves through disease or insect attack, prevents complete filling of the endosperm, the seed will be able to germinate, grow and reproduce the species for a further generation. The protein content of the seed coat, embryo and aleurone layer therefore tend to remain relatively constant (Doekes and Wennekes, 1982) with ‘surplus’ N being stored in endosperm proteins.

Using this conclusion, together with data on the distribution of protein between parts of the kernel (Table 1) we can calculate nitrogen uptake by wheat crops with different yield potentials and protein levels (Table 3). Grain nitrogen requirements double from 35 kg/ha to 70 kg/ha as the yield potential of a crop, with 10% protein in the grain, increases from 2 to 4 t/ha. If the protein content is increased to 12.5%, an additional 17.5 kg N/ha is required in the grain, assuming that all additional N ends up in storage proteins. Additional amounts of nitrogen which are not translocated from the straw to the grain must also be allowed for in the equation (about 5 kg N/ha/t grain, Table 3). Total nitrogen taken up by a crop yields 4 t/ha with 12.5% protein is therefore 107.5 kg. Other speakers at this Conference will point out that the efficiency with which plants are able to take up nitrogen from fertiliser sources is less than 100% so that minimum nitrogen requirements will be greater than those shown in Table 3.

Table 3. Calculated nitrogen uptake by wheat crops with the same grain size but differing in yield potential.

Whole grain (% protein)

Crop yield

(t/ha)

Grain N a (kg/ha)

Straw N (kg/ha)

Total N uptake
(kg/ha)

   

Bran

Germ

Flour

Total

   

10

4

7.0

2.8

25.2

35.0

10.0

45.0

 

4

10.5

4.2

37.8

52.5

15.0

67.5

 

4

14.0

5.6

50.4

70.0

20.0

90.0

12.5 c

4

14.0

5.6

67.9

87.5

20.0

107.5

a Assuming 17.5% N in grain protein and fractional distribution shown in Table 1

b Assuming harvest index 0.33 and 0.25% N in crop residues

c Achieved for example by late season N uptake by the crop. Note that only storage protein levels are increased (Doekes and Wennekes, 1982).

In practice, crop yields and grain protein contents respond in two quite different ways to nitrogen availability (Figure 2). In low fertility paddocks where there is little available nitrogen, small applications will produce large yield responses but grain protein percentage may decline. In other words the increase in the number of kernels is not matched by the increase in nitrogen so that the amount available to each kernel is reduced. A further increase in nitrogen availability will further increase yields and will also markedly increase grain protein content. Yields reach a plateau at high nitrogen availability while protein levels continue to increase with additional nitrogen.

Figure 2. Grain yield and protein responses to nitrogen availability

Conclusion

Low nitrogen availability is the most common limitation to protein synthesis. Grain proteins are synthesised only at the end of the growth cycle so that if the supply of nitrogen runs out, grain protein will be low. If there is sufficient nitrogen available for high yields, there will also be sufficient nitrogen available for acceptable grain protein levels.

References

1. Barlow, K.K., Buttrose, M.S., Simmonds, D.H. and Vesk, M. (1973). The nature of the starch - proteininterface in wheat endosperm. Cereal Chem. 50:443.

2. Bushuk, W. and Wrigley, C.W. (1974). Proteins: composition, structure and function. In ‘Wheat: Production and Utilization’, ed. G.E. Inglett, p.119. Avi, Westport, Conn.

3. Buttrose, M.S.(1963). Ultrastructure of the developing wheat endosperm. Aust.J.Biol.Sci. 16:305.

4. Doekes, G.J. and Wennekes, L.M.J. (1982). Effect of nitrogen fertilisation on quantity and composition of wheat flour protein. Cereal Chem. 59:276.

5. Duvick, D.N. (1961). Protein granules of maize endosperm cells. Cereal Chem. 38:374.

6. FAQ (1970). Amino acid content of foods and biological data on proteins.

7. FAQ nutritional studies no. 24. Food and Agricultural Organization of the

8. United Nations, Rome.

9. Fulcher, R.G., O’Brien, T.P. and Simmonds, O.H. (1972). Localization of arginine-rich proteins in mature seeds of some members of gramineae. Aust..J.Biol.Sci. 25:487.

10. Hinton, J.J.C. (1953). The distribution of protein in the maize kernel in comparison with that in wheat. Cereal Chem. 30:441.

11. Jennings, A.C. and Morton, R.K. (1963). Changes in carbohydrate, protein, and non-protein nitrogenous compounds of developing wheat grain. Aust.J. Biol.Sci. 16:318.

12. Kent, N.L. (1966). Subaleurone cells of high protein content. Cereal Chem. 43:585.

13. Konzak, C.F. (1977). Genetic control of the content, amino acid composition and processing properties of proteins in wheat. Adv.Genetics 19:407. Academic Press Inc., New York.

14. Lasztity, R. (1984). The chemistry of cereal proteins. CRC Press Inc., Florida, p.3.

15. Kasarda, D.D., Bernardin, J.E. and Nimmo, C.C. (1976). Wheat Proteins. In ‘Advances in Cereal Science and Technology’, ed. Y. Pomeranz, p.158. American Association of Cereal Chemists, St Paul, Minn.

16. MacMasters, M.M., Hinton, J.J.C. and Bradbury, D..(1971). Microscopic structure and composition of the wheat kernel. In ‘Wheat: Chemistry and Utilization’, ed. Y. Pomeranz, p.51. American Association of Cereal Chemists, St Paul, Minn.

17. Mitra, R. and Bhatia, CR. (1973). Studies on protein biosynthesis in developing wheat kernels. (Cited by Kasarda et al.. 1976).

18. Osborne, T.B. (1907). The Proteins of the Wheat Kernel. Carnegie Institute of Washington, Washington, DC. (Cited by Lasztity, 1984).

19. Pomeranz, Y., Carvajal, M.I., Hoseney, R.C. and Ward, A.B. (1970). Wheat germ in bread making. I. Composition of germ lipids and germ protein fractions. Cereal Chem. 47:373.

20. Simmonds, D.H.,.Barlow, K.K. and Wrigley, C.W. (1973). The biochemical basis of grain hardness in wh~at. Cereal Chem. 50:553.

21. Simmonds, D.H. (1978). Structure, composition and biochemistry of cereal grains. In ‘Cereals ‘78: Better Nutrition for the World’s Millions’, ed. Y. Pomeranz, p.105. American Association of Cereal Chemists, St Paul, Minn.

22. Simmonds, D.H. (1981). Wheat proteins: their chemistry and nutritional potential. In ‘Wheat Science - Today and Tomorrow’, eds, L.T. Evans and W.J. Peacock, p.149. Cambridge. Univ. Press.

23. Symes, K.J. (1969). Influence of a gene causing hardness on the milling and baking qualities of two wheats. Aust.J.Agric.Res. 20:971.

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